FEMS Microbiology Reviews 29 (2005) 231–262

www.fems-microbiology.org

The many faces of the helix-turn-helix domain:

Transcription regulation and beyond q
L. Aravind *, Vivek Anantharaman, Santhanam Balaji,
M. Mohan Babu, Lakshminarayan M. Iyer
National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Building 38A,
Room 5N50, Bethesda, MD 20894, USA

Received 20 November 2004; received in revised form 22 December 2004; accepted 23 December 2004

First published online 28 January 2005

Abstract

The helix-turn-helix (HTH) domain is a common denominator in basal and specific transcription factors from the three super-
kingdoms of life. At its core, the domain comprises of an open tri-helical bundle, which typically binds DNA with the 3rd helix.
Drawing on the wealth of data that has accumulated over two decades since the discovery of the domain, we present an overview
of the natural history of the HTH domain from the viewpoint of structural analysis and comparative genomics. In structural terms,
the HTH domains have developed several elaborations on the basic 3-helical core, such as the tetra-helical bundle, the winged-helix
and the ribbon-helix–helix type configurations. In functional terms, the HTH domains are present in the most prevalent transcrip-
tion factors of all prokaryotic genomes and some eukaryotic genomes. They have been recruited to a wide range of functions beyond
transcription regulation, which include DNA repair and replication, RNA metabolism and protein–protein interactions in diverse
signaling contexts. Beyond their basic role in mediating macromolecular interactions, the HTH domains have also been incorpo-
rated into the catalytic domains of diverse enzymes. We discuss the general domain architectural themes that have arisen amongst
the HTH domains as a result of their recruitment to these diverse functions. We present a natural classification, higher-order rela-
tionships and phyletic pattern analysis of all the major families of HTH domains. This reconstruction suggests that there were at
least 6–11 different HTH domains in the last universal common ancestor of all life forms, which covered much of the structural
diversity and part of the functional versatility of the extant representatives of this domain. In prokaryotes the total number of
HTH domains per genome shows a strong power-equation type scaling with the gene number per genome. However, the HTH
domains in two-component signaling pathways show a linear scaling with gene number, in contrast to the non-linear scaling of
HTH domains in single-component systems and sigma factors. These observations point to distinct evolutionary forces in the emer-
gence of different signaling systems with HTH transcription factors. The archaea and bacteria share a number of ancient families of
specific HTH transcription factors. However, they do not share any orthologous HTH proteins in the basal transcription apparatus.
This differential relationship of their basal and specific transcriptional machinery poses an apparent conundrum regarding the ori-
gins of their transcription apparatus.

2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

Keywords: Helix-turn-helix; DNA-binding; Transcription regulation; Evolution; Two-component systems; Structure

q
Edited by Mark J. Pallen.
*
Corresponding author. Tel.: +1 301 594 2445; fax: +1 301 435 7794.
E-mail address: [email protected] (L Aravind).

0168-6445/$22.00
2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.femsre.2004.12.008
232 L. Aravind et al. / FEMS Microbiology Reviews 29 (2005) 231–262

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 232
2. Structural scaffold of the HTH domain and its diverse elaborations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 233
2.1. HTH domains with a simple three-helical bundle and its extensions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 234
2.2. The winged HTH domain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 235
2.3. Other highly modified variants of the HTH domain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 236
3. General and specific aspects of the domain architectures of HTH proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 236
3.1. Simple architectures involving the HTH domain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 237
3.2. Combinations of HTH with other nucleic acid binding domains and protein–protein interaction domains. . . . . . 241
3.3. Combinations of the HTH domain with catalytic domains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 241
3.4. Architectures related to two-component, PTS and serine/threonine kinase signaling . . . . . . . . . . . . . . . . . . . . . 243
3.5. Architectures related to single-component signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 244
3.6. Unusual functional adaptations of the HTH domain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 245
4. The evolutionary classification of HTH domains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 246
4.1. Lineages of basic tri-helical HTH domains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 246
4.2. The tetra-helical HTH superclass and its derivatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 248
4.3. The wHTH superclass . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 249
4.4. Other miscellaneous families of HTH domains. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 252
5. Proteome-wide demographic trends of HTH domains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 252
6. General considerations on the natural history of the HTH fold and implications for the evolution of transcription. . . . 254
7. General conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 256
8. Supplementary material . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 257
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 257

1. Introduction teins, termed the specific transcription factors, were

found to belong to two distinct functional types: (1)
The general paradigms for the processes of transcrip- those which negatively regulated transcription of their
tion initiation and regulation first emerged from the pio- target gene (repressors) and (2) those which positively
neering studies on gene expression in bacteria and regulated transcription of their target genes (activators)
phages [1]. Transcription in bacteria was found to be [1]. The affinities of the specific transcription factors for
catalyzed by a single multi-subunit RNA polymerase, their target sequences on DNA were often found to be
which is recruited to promoters by means of a DNA- dependent on their interaction with low-molecular
binding protein, the sigma factor that recognizes specific weight compounds (effectors), which bound to them,
sequences upstream of genes [2–4]. The sigma factor and or phosphorylation and other post-transcriptional mod-
the RNA polymerase, together, constitute the basal ifications [1]. When transcription in eukaryotes was first
transcription apparatus that is required for the baseline investigated, several differences in the subunit composi-
transcription of all genes. Early studies, especially in the tion and architecture of the basal transcription machin-
Bacillus subtilis sporulation model, suggested that there ery and specific transcription factors were noted [7,8].
may be more than one sigma factor that might recruit However, the basic regulatory mechanisms in bacterial
the catalytic core of the RNA polymerase to alternative transcription, which were brought to light in early stud-
sets of genes. This provided a mechanism for regulating ies, remained applicable in a generic sense across the
the broad changes in gene expression, which correlate entire Tree of Life [1].
with the different developmental or differentiation states The pioneering investigations of Matthews, Ohlen-
of a bacterium [5,6]. A number of early studies on met- dorf, Sauer, Doolittle and co-workers in 1982 provided
abolic regulation in bacteria also indicated that there are the first glimpse of the features unifying diverse tran-
other regulatory DNA-binding proteins that act as scription regulators [9–13]. They showed that the phage
switches to control the expression of specific smaller sets lambda transcription regulators, cro and the cI repres-
of genes. These sets of genes are often collinear on the sor, and lacI, the lactose operon repressor, shared a sim-
chromosome, and encode components of a common ilar tri-helical DNA-binding domain. The 2nd and the
pathway for the utilization of a particular environmen- 3rd helices of this tri-helical domain constituted a
tal metabolite (e.g. lactose), or constitute interacting Helix-Turn-Helix motif, and this motif was shown to
components of a developmental pathway (e.g. lytic or be a critical determinant of their interaction with
lysogenic development of phages). These regulatory pro- DNA. Thus, these DNA-binding domains came to be
 L. Aravind et al. / FEMS Microbiology Reviews 29 (2005) 231–262 233

referred as the helix-turn-helix (HTH) domain. Se- may be deployed. The combined use of genome se-
quence and secondary structure analysis by these pio- quence data and high-throughput expression data also
neering workers suggested that the HTH domain was cast light on the multi-level transcriptional regulatory
a common DNA-binding motif found in several other networks in prokaryotes in which these HTH-containing
bacterial repressors as well as activators, such as the proteins functionally interact to maintain a particular
cAMP-dependent catabolite activator protein. They per- transcriptional state in the cell [42,43].
ceptively suggested that all these DNA-binding domains Over the years, the recruitment of the HTH domains
have descended from a common ancestor, through to biological functions beyond transcriptional regula-
duplication and divergence, thereby generating the tion has also become apparent. Some of these proteins,
diversity of transcription regulators that regulate bacte- participating in functions such as DNA repair and RNA
rial and phage genes [9,11]. Subsequent sequence analy- metabolism, exploit the nucleic acid-binding properties
sis revealed that DNA recognition by sigma factors was of the HTH just as in the case of transcriptional regula-
also mediated by HTH domains, similar to those ob- tion (for example see: [44–48]). However, there are other
served in the specific transcription factors [14–16]. In instances where the HTH may be adapted to very differ-
the second half of the 1980s the conserved domains of ent functions, such as mediating specific protein–protein
transcription factors regulating eukaryotic development interactions, or as a structural unit of a larger enzymatic
and differentiation, namely the homeodomains and Myb domain [49–51].
domains, were also noted to possess the HTH fold In this article we aim at providing a synthetic over-
[17,18]. These and other investigations suggested the view of the structural, functional and evolutionary
general significance of this module in DNA–protein diversity of the HTH domain from the vantage point
interactions across a wide phylogenetic spectrum of over two decades of intense investigation. We cur-
[18–21]. rently enjoy unprecedented advantages due to an enor-
An explosion of structural studies in the 1990s, while mous amount of genomic data, numerous high
strengthening the basic structure–function relation be- resolution structures, functional studies and sensitive
tween the HTH domain and DNA binding, also pro- computational tools to capture the highlights of the nat-
duced a large amount of data regarding the diversity ural history of HTH domains. Our focus is principally,
of DNA–protein interactions mediated by different ver- but not exclusively, on versions of the domain found
sions of the HTH domain. A number of sequence and in the prokaryotic super-kingdoms. We first discuss
structural analysis studies also uncovered HTH modules the structural diversity seen in this fold, followed by a
in several specific eukaryotic transcription factors, chro- discussion of the unifying themes in domain architec-
matin proteins like histone H1, and basal transcription tures of HTH proteins and their significance to different
factors such as TFIIB and TFIIE [22–30]. These findings biological functions. We next provide a higher-order
lead to the idea that the HTH domain is probably one of natural classification of these domains and discuss their
the most ancient conserved features of transcription genome-wide demography in light of the general adap-
apparatus, which was already present in the last univer- tive tendencies that can be inferred from comparative
sal common ancestor of all extant life forms (LUCA). genomics. In this context we also consider the adapta-
Studies during this period also showed that although ar- tion of the HTH to functional roles beyond transcrip-
chaea and eukaryotes possess a similar basal transcrip- tional regulation. Finally, we discuss the relevance of
tion machinery, the specific transcription factors of the the information gleaned from these diverse areas in
former are clearly closer to those of the bacteria than reconstructing the origin and evolution of transcription
the eukaryotes [30–34]. The other major development and its regulation.
in the later half of the 1990s was the birth of the genomic
era, unleashing the power of comparative genomics.
Starting with the earliest comparative genomic studies 2. Structural scaffold of the HTH domain and its diverse
it became apparent that the HTH domain was a highly elaborations
prevalent domain in prokaryotes [35]. Comparative
analysis also helped in identifying several major mono- The basic HTH domain is a simple fold comprised of
phyletic assemblages of HTH transcription factors, each three core helices that form a right-handed helical bun-
distinguished by their own distinctive sequence and dle with a partly open configuration. When it is dis-
structure features (for example see Refs. [36–41]). These played by placing the third helix in the front and in
classes often showed one or more distinctive domain the horizontal orientation, the 3 helices of the domain
architectures – i.e. the fusion of the HTH domain with form an approximately triangular outline (Fig. 1). We
additional globular domains in the same polypeptide. use this as the default orientation for all further illustra-
These globular domains, which are linked to the HTH tions and discussions. The characteristic sharp turn,
show a bewildering diversity, and point to the immense which is a defining feature of this domain, is situated be-
variety of functional contexts in which the HTH domain tween the 2nd and the 3rd helix, and typically does not
234 L. Aravind et al. / FEMS Microbiology Reviews 29 (2005) 231–262

have evolved to generate a more closed configuration

by interacting with the cleft (Fig. 1; see below for further
discussion). The 3rd helix, known as the recognition he-
lix, typically forms the principal DNA–protein interface
by inserting itself into the major groove of the DNA
[19,24,52]. Nevertheless, the individual residues involved
in DNA contacts may widely vary across the fold. Addi-
tional secondary contacts with DNA may be mediated
by other parts of the structure or even extensions outside
of the core HTH domain, such as a basic patch at the
N-terminus of helix-1 [52].
Despite the great sequence diversity observed in this
fold, there are a few sequence elements that are widely
conserved in members of the fold. The most character-
istic of these is the ‘‘shs’’ pattern (where ÔsÕ is a small
residue, most frequently glycine in the first position,
and ÔhÕ is a hydrophobic residue) that lies in the turn
between helix-2 and helix-3 of the core HTH structure.
The other well-conserved signatures is the ‘‘phs’’
(where ÔpÕ is a charged residue, most frequently gluta-
mate) that is present in helix-2 (Fig. 1). The conserved
hydrophobic residues in these motifs, together with at
least two other conserved-hydrophobic residues seen
in helix-1 and helix-3 localize to the interior and form
the characteristic hydrophobic core that stabilizes the
domain (Fig. 1). The conservation of these elements
across diverse members of this fold from the 3 super-
kingdoms of life, taken together with the conserved
structure–function associations of this fold strongly
supports the monophyletic origin of HTH domains
[9,11] from a common ancestor that bore the above-
Fig. 1. Salient structural and features of the HTH domains and its
chief variants. Helices are shown in green and labeled with an ÔHÕ,
mentioned sequence features.
strands are in blue. The top panel shows a representative of a simple Based on their distinctive features, members of the
tri-helical HTH domain (PDB: 1k78). Residues that are strongly HTH fold can be divided into two major structural clas-
conserved across all HTH domains are shown in the top panel in stick ses and a few other highly derived structural classes that
representation. On the right a surface view of the same domain with contain drastic alterations of the core HTH domain.
the shallow cleft, typical of the partly ‘‘open’’ configuration of the tri-
helical bundle, is shown using the surface view. In the middle panel a
representative of the tetra-helical bundle (PDB: 1a.4) is shown to the
left, while the metal-chelating HTH domain of the retroviral integrases 2.1. HTH domains with a simple three-helical bundle and
is show to the right (HIV integrase, PDB: 1k6y). In the bottom panel a its extensions
simple 2-stranded winged HTH (pdb: 1smt) is shown to the left, while a
4-stranded winged HTH (PDB: 1cgp) is shown to the right.
The simplest version of the HTH domain, the basic
tri-helical version, is comprised entirely of the three core
tolerate insertions or distortions. However, the loop be- helices with no additional elaborations (Figs. 1 and 2).
tween helix-1 and helix-2 shows far greater variability This configuration appears to be closest to the ancestral
and may accommodate several modifications in the dif- state of the domain and is widely seen across the three
ferent classes of HTH domains. Furthermore, diverse N- super-kingdoms of life. This version is represented by
and C-terminal extensions to the core tri-helical bundle the HTH domains such as the region-3 and region-4 of
are also encountered in several classes of HTH domains. the sigma factors, the RPB10 subunit of the DNA-
The partly open configuration of the bundle results in a dependent-RNA polymerases of the archaeo-eukaryotic
shallow cleft between helix-3 and helix-1 on the side lineage, bacterial transcription factors of the FIS family,
opposite to helix-2. This cleft appears to have acted as the eukaryotic Homeo, POU and Myb domains, the
a structural niche that favored the evolution of addi- eukaryotic BRCA2 tower domain, and the paired mod-
tional structural elements to pack into it via hydropho- ule and related HTH domains of transposases and resol-
bic interactions. Thus, most of the extension to the core vases (Fig. 2). A distorted version of the basic tri-helical
HTH domain are structural elements that appear to HTH domain is also seen in the zinc-chelating domain
 L. Aravind et al. / FEMS Microbiology Reviews 29 (2005) 231–262 235

Fig. 2. A pathway showing the structural elaboration of the simple HTH domain into its diverse versions. Strands are shown as yellow arrows with
the arrow heads at the C-terminus, helices are shown as blue cylinders. The orange arrows show the probable routes of transformation of the HTH
fold. The two possible origins of the L11 like HTHs are shown by dotted arrows with question marks. The topologies have been constructed using the
following PDB enteries: (1) simple trihelical bundle: 2hdd (2) FF domain: 1h40 (3) Tetrahelical bundle: 1d5y (4) Multihelical bundle: 1ais (5) MetJ/
Arc: 2cpg (6) MerR: 1jbg (7) L11: 1mms (8) CRP-like: 1cgp (9) T. vaginalis initiator: 1pp7 (10) 3 stranded wHTH: 2dtr (11) Methionine
aminopeptidase: 1xgs (12) winged HTH: 1i1g (13) wHTH with a C-terminal helix: 1jgs.

of several retroviral integrases [53], in which helix-3 is [55], is another more divergent version of the TFIIB-like
packed more closely against the other helices by means HTH domains. This version of the fold is very infre-
of a Zn ion chelated by conserved cysteines and histi- quent in the bacteria, and is represented by a circularly
dines at the ends of helix-1 and helix-3 (Fig. 1). Like- permuted version seen in the sporulation regulator
wise, in RPB10, a set of zinc-chelating cysteines help Spo0A from spore-forming Gram-positive bacteria
in stabilizing an N-terminal loop against helix-3 [54]. [56]. Other versions, which represent relatively infre-
The tetra-helical version of HTH domain is an elabo- quent elaborations of the basic tri-helical version, are
ration of the basic tri-helical version and is characterized the KorB-like HTHs [57] and the FlhD-like HTHs
by an additional C-terminal helix which packs against [58]. The former version is characterized by an addi-
the shallow cleft formed due to the open configuration tional N-terminal helix that packs against the basic 3-he-
of the tri-helical core (Figs. 1 and 2). Several major fam- lix core and the later contains a C-terminal helical
ilies of prokaryotic transcription factors display this ver- extension that packs very differently from the helical
sion of the domain. The multi-helical version, typified by extension seen in the above-mentioned classical tetra-
the archaeo-eukaryotic basal transcription factor helical forms (Fig. 2).
TFIIB, is a further elaboration of the tetra-helical
HTH, wherein two additional helices have been added 2.2. The winged HTH domain
to the N-terminus of the tetra-helical core, resulting in
a larger globular helical bundle (Fig. 2). The Bright The winged HTH (wHTH) domains are distinguished
(ARID) domain, a eukaryotic DNA-binding domain by presence of a C-terminal b-strand hairpin unit (the
236 L. Aravind et al. / FEMS Microbiology Reviews 29 (2005) 231–262

wing) that packs against the shallow cleft of the partially together with the notable structural and sequence simi-
open tri-helical core [24,25]. The simplest versions of the larities with the HTH domains, suggest that the RHH
wHTH domains contain a tight helical core similar to domain was derived from the HTH domain through
basic tri-helical version followed by the two-strand hair- conversion of the N-terminal helix to a strand. Concom-
pin (Figs. 1 and 2). However, many wHTH domains itant with this modification, the N-terminal strand,
display further serial elaborations of the b-sheet. In which came to lie atop the recognition helix, appears
the 3-stranded version, the loop between helix-1 and he- to have taken up the principal DNA-binding role of this
lix-2 assumes an extended configuration and is incorpo- protein.
rated as the 3rd strand in the sheet, via hydrogen The DNA-binding domain of the bacterial transcrip-
bonding with the basic C-terminal hairpin (Fig. 2). In tion regulator MerR defines an aberrant derivative of a
the 4-stranded version, the linker between helix-1 and 3-stranded version of the wHTH domain, in which he-
helix-2 also forms a hairpin with two b-strands, and lix-1 has been lost (Fig. 2). In addition to the MerR fam-
along with the C-terminal wing forms an extended b- ily of bacterial repressors this version of the fold is seen
sheet (Fig. 2). In versions that bind nucleic acids, the in a wide variety of phage, bacterial and eukaryotic
wing often provides an additional interface for substrate DNA-binding proteins and translation factors. The
contact, typically by interacting with the minor groove topoisomerase II family has two copies of wHTH do-
of DNA through charged residues in the hairpin mains [63]. One of these wHTH domains contains a
[24,25,27]. The two- and three-stranded versions of the large insert between helix-1 and helix-2. This insert con-
wHTH are encountered in DNA-binding domains of tains an extended b-sheet structure that forms a brace
some of the largest families of prokaryotic transcription around double-stranded DNA. Also present in this in-
factors, as well as several eukaryotic DNA-binding do- sert is a second wHTH domain which is circularly per-
mains. The single-strand RNA-binding La domains also muted with helix-1 occurring to the C-terminus of the
have a version of the wHTH fold with a slightly ex- wing. The two structurally related ribosomal proteins
tended and variable insert between helix-1 and helix-2. L11 and S18 represent another distinctive derivative of
An unusual version of the 4-stranded wHTH domain, wHTH domain. While they share a b-strand hairpin be-
with an additional small helical insert after helix-1, tween helix-1 and helix-2 with the 4-stranded wHTH,
is observed in the orphan transcription-initiator- unlike the latter they possess only a single C-terminal
sequence-binding protein from the parabasalid protist, strand (Fig. 2). A highly modified HTH domain seen
Trichomonas vaginalis [59] (Fig. 2). The Fur family of in the catalytic domain of the phage integrases shows
bacterial transcription factors, which is involved in me- a rare insertion between helix-2 and helix-3 of the core
tal-responsive transcriptional regulation, shows a regu- domain [63]; however, this insert does not distort the
lar 2/3-stranded wHTH domain, but the wing is structure of the core. The FF-domain [64] and the C-ter-
incorporated into a large sheet formed with additional minal domain of the PP2C protein phosphatase [65] de-
C-terminal strands. A circularly permuted version of fine another highly modified version of the HTH
the basic wHTH domain, with one of the strands of domain that is currently only known from eukaryotes.
the wing moved to the N-terminus, is seen in the C-ter- This version domain shows the insertion of a helical in-
minal accessory domain of the methionine aminopepti- sert between helix-1 and helix-2 resulting a different
dase-2 (Fig. 2) [60]. packing of the elements and a distortion of the fold
(Fig. 2).
2.3. Other highly modified variants of the HTH domain

The MetJ-Arc family (also known as ribbon-helix-

helix/RHH family) of transcription factors is, to date, 3. General and specific aspects of the domain architectures
known only from the prokaryotes. They are obligate di- of HTH proteins
mers, which pair through a single N-terminal strand,
and possess a C-terminal helix-turn-helix unit (Fig. 2). HTH domains are combined with other domains in
The organization of the C-terminal helical unit is identi- the same protein giving rise to an astonishing array of
cal to corresponding unit in the classical versions of the domain architectures (Table 1 and Fig. 3). Despite the
HTH domain, and it shows the characteristic conserved diversity, all the architectures can be classified into a
sequence features of the HTH domain [61]. The sheet small number of generic architectural classes, the mem-
formed by the N-terminal strands of the domain is in- bers of each class being unified by certain general orga-
serted into the major groove of DNA [61]. Mutagenesis nizational and functional principles. These generic
experiments have shown that even single mutations in architectural classes illustrate how natural selection
the N-terminal strand convert the strand of the RHH has convergently engineered similar functional solutions
domain to a helix, and result in a structural packing that using a relatively small repertoire of domains [66].
is closer to the canonical HTH domain [62]. This result, Hence, the more highly populated architectural classes
 L. Aravind et al. / FEMS Microbiology Reviews 29 (2005) 231–262 237

Fig. 3. Examples of domain architectures of proteins with HTH domains. The 3 panels show the HTH domains in one component systems, those in
two component systems and those fused to enzymatic domains. Below each protein the name of the protein or its family is indicated. The domain
names and abbreviations are as shown in the Table 1. Additional abbreviations are H – any HTH domain; wH – winged HTH; Assd – archaeal
specific signaling domain; Acyclase – adenylyl cyclases; AATRS – aminoacyl tRNA synthetase; TPR – tetratricopeptide repeats; HAMP – domain
present in histidine kinases, adenylyl cyclases, methyl-accepting proteins and phosphatases, chro-chromodomain BTAD- conserved domain found in
bacterial signaling proteins, CPS-D1, the domain 1 of the large subunit of the carbamoylphosphate synthetases, MGS- the methylglyoxal synthase
domain that binds ornithine in the carbamoylphosphate synthetases, ParB- the OB-fold nuclease domain seen in ParB proteins, B-hel- a conserved
beta-barrel seen in the Lhr helicases. The transmembrane regions are indicated by yellow boxes. The grey boxes represent uncharacterized globular
domains that are unique to a particular family of proteins. Af: Archaeoglobus fulgidus, Ana: Nostoc sp., Blic: Bacillus licheniformis, Bs: Bacillus
subtilis, Bthe: Bacteroides thetaiotaomicron, Ec: Escherichia coli, Llac: Lactococcus lactis, Lmon: Listeria monocytogenes, Mjan: Methanococcus
jannaschii, Mlo: Mesorhizobium loti, Mmaz: Methanosarcina mazei, Mcap: Methylococcus capsulatus, Mthe: Methanothermobacter thermautotro-
phicus, Mtu: Mycobacterium tuberculosis, Phor: Pyrococcus horikoshii, Poke: Planomicrobium okeanokoites, Tm: Thermotoga maritima, Sent:
Salmonella enterica, Spyo: Streptococcus pyogenes, Hs: Homo sapiens, Sc: Saccharomyces cerevisiae.

are likely to represent the more successful functional machinery. The eukaryotic ribosomal protein S10 and
solutions in which the HTH domain has been involved. the histone H1 respectively contain RNA and DNA
binding versions of the wHTH domain, which is fused
to low-complexity sequence that forms a non-globular
3.1. Simple architectures involving the HTH domain tail [67]. Such non-globular extensions that play a role
in non-specific nucleic acid contacts and protein con-
The simplest architectures involving the HTH do- tacts during transcription activation are very common
main are seen in certain proteins related to the cI repres- in eukaryotic transcription factors with HTH domains
sors (e.g. the archaeal repressors typified by AF1793), [41]. A family of bacterial proteins typified by the B. sub-
most proteins of the MetJ-Arc superfamily and the Fis tilis sigma D regulator YlxL (SwrB) [68,69] contains a
proteins. These proteins are almost entirely comprised HTH domain fused to a N-terminal transmembrane re-
of just a standalone HTH, and might, at best, have some gion (Fig. 3). These HTH proteins might regulate tran-
small extensions that play a role in dimerization or inter- scription under the influence of signaling events
actions with other components of the transcriptional associated with the cell membrane. The next level of
 238
Table 1
Globular domains frequently linked to the HTH domain in the same polypeptide
Domain Structure Placement of HTHa Comments
Domains in two-component, phosphorelay and S/T kinase signaling cascades
REC (receiver domain) a/b. [Flavodoxin-like topology]. 1.19N; 78.35C Is phosphorylated on an aspartate residue by the histidine kinase dependent
PDB 1NTR phospho-relay system. Typically found fused to OmpR and LuxR-family
(in NarL-like proteins) HTH domains
Histidine kinase a + b. PDB 1BXD 0.10N; 0.03C Usually occurs as a standalone subunit, but on few instances is fused to the
downstream transcription factor with receiver and HTH domains
3H a + b fold 1.00N A domain sharing a common fold with the HPr domain of the PTS system. It has 3
conserved histidine residues that are likely to be phosphorylated
FHA b fold PDB 1LGQ 0.23N; 0.16C A phosphoserine/threonine peptide-binding domain. Combinations with HTHs are
expanded mainly in Actinomyctes, which have numerous serine/threonine kinases

L. Aravind et al. / FEMS Microbiology Reviews 29 (2005) 231–262

HPT (histidine phosphotransfer a helical domain PDB 1QSP – Receives a phosphate on a conserved histidine in the two-component relay. Found
domain) fused to OmpR family wHTH domains along with Receiver domains in
cyanobacteria
PRD (PTS Regulatory Domain) a helical domain PDB 1H99 3.84N Typically phosphorylated by the EIIB and HPr. It is often found fused to the HTH
in the LevR-subfamily with the NtrC-type AAA+ ATPases
PTSIIA a+b – A conserved domain of the PTS system that receives the phosphate in the
phosphor-relay and regulates the sugar permeases
PTSIIB a + b PDB 1BLE – A conserved domain of the PTS system that receives the phosphate in the
phospho-relay and phosphorylates the PRD domain
SMBDs typically associated with one-component systems
ACT (aspartokinase, chorismate a + b sandwich [RRM-like fold]. 0.19N Binds various small molecule ligands such as amino acids and purine derivatives
mutase, TyrA domain) PDB 1PSD
Lrp-C a + b sandwich [RRM-like fold] 15.55N This domain is exclusively found at the C-termini of proteins of the Lrp family and
is closely related to the ACT fold. It is likely to be an amino-acid-sensing derivative
of the ACT fold which is unique to the Lrp proteins.
ArgR-C a + b. DcoH fold. PDB 1XXA 3.35N The amino acid-sensing domain exclusively found fused the ArgR-family of HTH
domains
DSBH (Double-stranded A double-stranded helix of 6.45N; 14.68C A widespread fold with numerous enzymes that typically function as oxidases or
b-helix domain) strands. PDB: 2ARC. oxygenases. Most members of this fold contain 3 conserved residues that play a role
in ligand-recognition, as well as catalysis in the enzymatic versions of the fold. Binds
carbohydrates, oxalate and amino acids
Boa1-N All b – Domain found at the N-terminus of the Boa1-like archaeal HTHs that may be
distantly related to the DSBH domain
AlcR-N Two distinct sub-structures with 1.65C This domain shows an N-terminal all b sub-structure that is likely to form a DSBH
all b and all a sub-structures structure and a C-terminal all-a unit (Fig. 6). A lineage specific expansion is seen in
Nostoc sp. (17 proteins). Many versions contain a conserved cysteine which may be
the site for ligand-interaction or attachment of a prosthetic group
cNMPBD (cNMP-binding A double-stranded b-barrel 7.65 C Binds 3 0 -5 0 cyclic nucleotides and typically found fused to the C-terminus of the
domain) domain. PDB: 1RGS Crp-like HTH domains
CBS (Cystathionine a/b; in nearly all cases, a dimer. 2.61N A ligand-binding domain that probably binds a wide range of ligands which may
beta-synthase) PDB 1ZFJ include purine nucleotides and cyclic nucleotides. It is found fused in different
proteins to both wHTH and cI-like tetra-helical HTH domains
UTRA (UbiC, transcription a + b. PDB 1FW9 13.39N Versions of this domain appear to accommodate a wide range of ligands that
regulator associated domain) include amino acids, sugars, fatty acids and alkylphosphonate. Always found fused
to a HTH domains of the HutC/FarR subfamily of the GntR family. Shares a fold
with the bacterial Chorismate Lyases
DeoR-C a/b PDB 1LK7 0.48N This domain shares a common fold with the phosphosugar isomerases such as D-
ribose-5-phosphate isomerase. It acts as the sugar-binding domain of the DeoR
family of transcription factors
DtxR-N a-helical domain followed by 3.90N This domain is found exclusively in the iron sensors of the DtxR-Fur family of HTH
SH3-like barrel. PDB 2DTR regulators
FadR-C a-helical. PDB 1H9G 23.58 N; 0.35C A unique seven-helical fold found fused to the subfamily of the GntR family typified
by the FadR protein
PBP-I (periplasmic binding a/b PDB: 1JWL 29.35N; 1.35C Members of this fold bind diverse small molecule ligands. Usually found in the
protein type-I domain) N-termini of the LacI family
PBP-II (periplasmic binding a/b PDB: 1AL3 87.97N; 0.61C Members of this fold bind diverse ligands like N-acetylserine, thiosulphate, amino
protein type-II domain) acids, carbohydrate. It is usually found in the C terminus of LysR family
T-OB All b PDB: 1B9M 1.23N A modified version of the OB fold that typically functions as an obligate dimer.
Found in ModE family where it binds molybdate
PAS (Per-Arnt-Sim domain) a/b. PAS-like fold PDB: 2PYP 13.61N; 3.48C A widely utilized superfamily of ligand-binding domains that bind a range of ligands

L. Aravind et al. / FEMS Microbiology Reviews 29 (2005) 231–262

such as heme, Flavin nucleotides, cinnamate. The ligand-binding domains of the
IclR family are divergent versions of the PAS domain
GAF (cGMP phospho- a/b. PAS-like fold 0.16N; 2.10C Another widely utilized superfamily family of ligand-binding domains sharing a
diesterase, Adenylate cyclase, common fold with the PAS domain. Binds ligands such as cNMPs, tetrapyrroles
FhlA domain) and formate. Often found at the N-terminus of NtrC-like proteins
TraR-N a/b. PAS-like fold. PDB: 1L3L 1.90C N-terminal domain of Agrobacterium TraR transcription factor. It is always found
at the C-terminus of several transcription factors of the LuxR family
PocR Predicted PAS-like fold 0.23C Domain found fused to the AraC family HTH domains in the 1,2-propanediol-
dependent transcription. It also occurs in other contexts fused to diverse signaling
domains. Secondary structure predictions suggest that it adopts a PAS-like fold
NTF2 a+b – A domain found in several enzymes like Steroid isomerases, Scytalone dehydratase
and the carotenoid binding protein. Likely to function as a SMBD fused to certain
sigma factors
GyrI a + b (contains a duplication of 4.55N Frequently found associated with HTH domains of the MerR family. The
SHS2 modules) drugbinding domain of BmrR

Enzymatic domains
NadR nucleotidyl transferase a/b. HUP fold; PDB 1LW7 0.48N The catalytic domain functions in the adenylation of the nicotinamide
domain (HIGH) mononucleotide in NAD biosynthesis
mlr6529-C module with Contains two distinct sub- 2.16N A novel conserved module typically found at the C-terminus of HTH domains of
metalloprotease-like and domains An N-terminal all a-unit the cI-like family. The N-terminal sub-domain possesses the same fold of as the
metal-chelating domains and a C-terminal a + b unit that Zn-dependent metalloproteases but many copies of it may be catalytically
might adopt a PAS-like fold inactive as they show disruptions of HEXXH signature. The C-terminal sub-
domain contains a conserved group of 4 cysteines, which suggests that it chelates
metals. Its predicted secondary structure suggests that it may adopt a PAS-like
fold
Biotin ligase domain BirM (Fold: Class II aaRS and 2.65N The ligase domain activates biotin to form biotinyl-5 0 -adenylate which acts as an
biotin synthetases) and BirC effector for transcriptional repression. Its regular activity is the transfer of biotin
(Fold: SH3-like barrel) PDB moiety to biotin-accepting proteins
1HXD
PLPDE PDB 1DJU 9.48N Pyridoxal phosphate-dependent aminotransferases. They are typically found fused
to GntR family HTH domains
Sugar isomerase (SIS) a/b PDB 1M3S 2.39N Usually associated with regulators of polysaccharide metabolism regulons
Uroporphyrinogen-III synthase a+b 12.65N An enzyme in Porphyrin biosynthesis pathway. Usually found fused to a wHTH
domain of the OmpR family

239
(continued on next page)
 240
Table 1 (continued)
Domain Structure Placement of HTHa Comments
Phosphoribosyltransferase a/b PDB 1STO 0.77N These enzymes transfer PRPP an activated form of phosphoribose to orotate and
(PRTase) purines in nucleotide biosynthesis. Independent fusions of HTHs to orotate and
purine phosphoribosyltransferases are seen
Sugar kinase a/b. RNAse H fold 6.77N Sugar kinases involved in polysaccharide metabolism are usually found fused to a
distinct subfamily of wHTH domains of the MarR family
b-D-Xylosidase a/b (TIM barrel) PDB: 1UHVD 0.58N This enzyme is found fused to the AraC-like HTH domains in regulators of
polysaccharide metabolism in low GC Gram positive bacteria
MJ0056 a+b 0.39N Found fused to HTH domain in well-conserved subfamily of MarR transcription

L. Aravind et al. / FEMS Microbiology Reviews 29 (2005) 231–262

factors that are restricted to the archaea. Genes encoding these proteins are often
found in a conserved gene-neighborhood with enzymes of the riboflavin
biosynthesis pathway. The conservation pattern suggests that this domain is also
enzymatic and may be a novel diaminohydroxyphosphoribosylaminopyrimidine
deaminase, because a conventional enzyme has not been identified in most archaea
V4R (Vinyl-4 reductase domain) a+b – This domain might bind intermediates of chlorophyll or porphyrin biosynthesis
pathways
Thioesterase domain a+b – A thioesterase domain proto-typed by the 4-hydroxybenzoyl-CoA thioesterase. This
domain is found fused to HTH domains of the GntR family in several proteins from
low-GC Gram positive bacteria. The proteins usually also possess CBS domains
Cyanate lyase a+b – Always found fused to a cI-like HTH domain. The enzyme detoxifies cyanide by
combining it with bicarbonate to produce ammonia and carbon-dioxide
ThiJ_PfpI a/b PDB: 1OI4 3.97C Domains of this superfamily possess a diverse range of activities, such as protease,
catalase, 5 0 -phosphoribosylformylglycinamide:L-glutamine amido-ligase, and
redox-dependent molecular chaperone activity. Usually they are found fused to the
AraC family HTH domains
LexA Protease domain All b 6.71N Serine protease domains of the signal peptidase fold. They are found fused to both
cI-like and wHTH domains
Carbamoyl phosphate synthatase a/b fold. PDB 1a9x – A dyad of HTH domains are found between the carboxy and carbamate
phosphorylating units of the large subunit of enzyme
PQQ biosynthesis protein C a helical. PDB 1RCW – Iron-binding redox enzyme involved in biosynthesis of Coenzyme Pyrroloquinoline
domain quinone. Fusions to HTH seen only in cyanobacteria
S-adenosyl-L-methionine- a/b. Rossmann fold 1.55N; 0.26C There have been multiple independent fusions to methyltransferases with different
dependent methyltransferase substrate specificities. These include fusions of the ArsR family (Pseudomonas
domain PA0547) and MerR (Bacillus BC2672) families with methylases of unknown
specificities, plant isoflavone O-methyltransferases and restriction methylases
Methyl-DNA protein a/b. A truncated RNAse H fold 8.52C These enzymes transfer alkyl groups from O-6-methylguanine-DNA to themselves.
methyltransferase The methyl acceptor is a conserved cysteine in the wing of the wHTH domain
AlkA a + b, TBP-like domain followed 0.48N An a-helical DNA glycosylase involved in DNA repair. The HTH domains in these
by helical HhH domain. PDB proteins belong to the AraC family
1DIZ
Ribonuclease R (RnaseR) a+b 3.06N A multi-domain enzyme involved in the processing of tmRNA
P-Loop NTPase a/b. P-loop fold 12.10N; 51.97C Diverse versions of the P-loop fold are fused to the HTH domain. These include the
AAA+ superclass members like Cdc6, MCM and DnaA, STAND NTPases in the
AfsR and MalT like regulators and GTPases in the case of the SelB and IF-12
proteins
DOC Predicted a-helical 1.65C A metal dependent enzymatic domain predicted to function as a nuclease
 L. Aravind et al. / FEMS Microbiology Reviews 29 (2005) 231–262 241

architectural diversification involves tandem duplica-

Domains possessing the same fold as the TolB-N terminal domain are fused to HTH

The number in this columns gives the frequency (per 1000 HTH proteins in prokaryotic genomes) of HTH domains located immediately adjacent to the globular domain under discussion. The
Catalytic domain found in topoisomerases, primases and nucleases with a catalytic

domains like IagA from S. typhi. This domain is invariably followed by a further set

A domain often found fused to a P-loop ATPase in the animal schalfen proteins and
A nuclease domain of the OB-fold involved in plasmid partitioning. Always found

These domains are involved in both DNA binding and protein-protein interactions.

ÔNÕ indicates that the HTH occurs N-terminal to the domain under discussion, whereas C indicates that it occurs to the C-terminus of the domain under discussion. A total set of 31100 proteins from
between the schlafen domain and the wHTH domain. Operon architectures suggest

184 prokaryotic genomes were analyzed. A Ô–Ô indicates that the HTH domain is either not found immediately adjacent to the module under discussion or that such combinations are very infrequent.
Two related RNA-binding domains typified by the bacterial ribosomal S1 protein
They are common in the archaeo-eukaryotic lineage in HTH proteins like TFIIE,

wHTH domain is typically accompanied a second ‘‘middle domain’’ that occurs

tions of HTH domains. Such versions are encountered

GTPases in some archaeo-eukaryotic proteins. The version associated with the

that they may be subunits of restriction-modification or DNA repair systems

in the sigma factors, where regions 3 and 4 correspond
to a tandem duplication of HTH domains [70]. The
Myb/SANT family of HTH domains in eukaryotic chro-
matin proteins and transcription factors also tend to
show multiple tandem copies [71]. In certain cases, such
duplications of the HTH domain in the same protein are
accompanied by functional diversification of the copies.
in association with HTH domains of the KorB family

For example, in the sigma factors, the HTH domain cor-

responding to the region-3 is involved in contacting the
core RNA polymerase complex, while the HTH domain
associated with region-4 binds DNA associated with the
-35 element of the promoter [70].
and the cold shock protein (CSP)
DxD motif at the active site

3.2. Combinations of HTH with other nucleic acid binding

domains and protein–protein interaction domains
MBF1 and TFIIB

of TPR domains

HTH domains are occasionally combined with other

domains that interact with macromolecules resulting in
multivalent polypeptides. Examples of such architec-
tures are the basal transcription factors, MBF1 and
TFIIE, of the archaeo-eukaryotic lineage, which com-
bine the HTH with a rubredoxin-like Zn-ribbon domain
[30]. The Zn-ribbon could either function as a second
potential nucleic acid binding domain or mediate spe-
cific protein–protein interactions with the basal tran-
scriptional machinery. Similarly, combinations of the
HTH with another four-helical DNA binding-domain
are seen in the plasmid encoded KorB proteins [57]. In
3.87C

the eukaryotes, the HTH is fused to the single-strand

DNA-binding OB-fold domains replication factor RP-
–
–

A, while in the tumor-suppressor protein, BRCA2, a basic

tri-helical HTH is inserted within one of its three OB-
folds [72]. In a class of widespread bacterial proteins,
prototyped by YitL, a wHTH domain is fused with
N-terminal domain is a + b

RNA-binding S1 domains, suggesting that this group

of proteins may perform an as yet uncharacterized role
All b. PDB 1VZ0

in bacterial RNA metabolism (Fig. 3). The domain

Metal Chelating

All b; OB fold

architectures suggest that these multi-domain proteins

usually function as adaptors that may bridge two mac-
romolecular complexes, such as specific transcription
a+b

factors and the basal transcriptional machinery, by

a/b

way of the multiple interaction surfaces provided by

their distinct domains. An alternative general function
for these proteins, which is not mutually exclusive of
Schalfen N-terminal and middle

the previous one, is to induce conformational changes

in nucleic acids by binding them at multiple sites.
Miscellaneous domains
Rubredoxin-like ZnR

3.3. Combinations of the HTH domain with catalytic

S1 and S1-like

domains
domains
TOPRIM

TolB-N

The HTH domain is frequently combined with a

ParB

diverse set of catalytic domains, and there are several

242 L. Aravind et al. / FEMS Microbiology Reviews 29 (2005) 231–262

general functional trends associated with such combina- In related examples, the HTH domain recruits a cat-
tions. One common association of the HTH domain alytic domain that may act on proteins, rather than nu-
with catalytic domains represents the utilization of the cleic acids. A striking example of this is the wHTH
domain as a substrate-recognition or localization do- domain fused to the N-terminus of the Rio family of
main. The HTH is observed to be linked to catalytic do- protein kinases from archaea and eukaryotes [30]. The
mains in several proteins involved in DNA replication Rio family of kinases function in 40S ribosomal subunit
(e.g. the FtsK–HerA superfamily ATPase domain [73] maturation [80,81], and the wHTH domain recruits the
in bacterial chromosome pumping protein FtsK [74], linked protein kinase domain to an rRNA processing
or the AAA+ ATPase domain in MCMs and Cdc6/ protein complex. The LexA protein, the repressor of sev-
ORC1, which function in archaeal and eukaryotic repli- eral bacterial DNA repair genes, represents another var-
cation initiation [75]) and repair (e.g. AlkA-type helical iation on this general functional theme. It contains a
DNA glycosylase domain), certain restriction endonu- protease domain of the signal peptidase fold fused to a
cleases (e.g. FokI [45]) and modification methylases wHTH domain. The protease domain catalyzes an auto-
(e.g. ScrFIA methylase) (Fig. 3). In course of this survey catalytic cleavage in response to a DNA damage signal
we noted that the DprA protein (Smf/Dal/CilB), which and triggers dissociation of its wHTH domain from tar-
is required for protecting DNA during transformation get sequences, thereby allowing transcription of DNA
in several bacteria [76], contains a wHTH domain fused repair genes [82]. Architectures analogous to LexA are
to the C-terminus of a large globular domain containing also seen in the repressors typified by the heat-response
a specialized version of the Rossmann fold (Fig. 3). This transcription factor HdiR from the Lactococcus lactis
wHTH domain might recruit the Rossmann fold do- [83], where a LexA-like protease domain is fused to a
main to DNA, and enable it to catalyze an as yet cI-like HTH instead of the wHTH seen in LexA (Fig.
uncharacterized DNA-modifying activity that is re- 3). This implies that the mechanism of transcription reg-
quired for efficient transformation. Most transposases ulation with a proteolytic processing step was innovated
and integrases of diverse mobile DNA elements have independently on at least two occasions in evolution.
at least one HTH domains that help their catalytic do- The next major architectural theme involving combi-
mains associate with DNA [77]. In these instances the nations of HTH and enzymatic domains appears to be
HTH either serves as an additional tether that recruits related to feedback regulation of metabolic pathways.
the catalytic domain to DNA or it participates in sub- In such combinations, the HTH domain is fused to an
strate recognition. An extreme case of this functional enzymatic domain catalyzing a key step in a biosynthetic
theme is seen in the topoisomerases: the HTH domains pathway, and usually regulates the transcription of
have supplied, on at least four independent occasions, genes in that pathway. One of the archetypal representa-
the catalytic tyrosine of these enzymes that is covalently tives of this architectural theme is the biotin operon
linked to DNA ([63,78] and LA unpublished). Similarly, repressor, BirA, which contains an N-terminal HTH do-
in the methyl-DNA protein methyltransferase (O-6- main fused to a C-terminal biotin ligase domain [23]. In
methylguanine-DNA alkyltransferase), a wHTH do- the presence of biotin the enzymatic domain synthesizes
main fused to a truncated domain of the RnaseH fold the co-repressor, and the HTH domain represses the
bears the cysteine that receives the alkyl group from transcription of the biotin biosynthesis genes. Compara-
damaged DNA [46]. Continuing on this theme, it has tive genomics suggests that architectures involving fu-
been previously noted that the catalytic domain of the sions to a range of enzymes from cofactor, nucleotide,
lambda integrase family has itself evolved from a dupli- amino acid and carbohydrate metabolism are fairly
cation of the HTH domains [63]. In some cases, the common in archaea and bacteria [30] (Table 1). Some
HTH domain has also been recruited by enzymes in- notable fusions include combination of the HTH with
volved in RNA metabolism and translation for binding nicotinamide mononucleotide adenylyl transferase and
RNA, especially double-stranded structures peculiar to a P-loop kinase in NadR, with the pyridoxal-phosphate
certain RNAs. For example, the GTPase module of dependent aminotransferase domain (in bacterial HTH
the bacterial selenocysteine-specific elongation factor proteins of the GntR family), the orotate phosphoribo-
(SelB) is recruited to a specific RNA hairpin of seleno- syltransferase (in archaea), sugar kinases (Rok family in
protein encoding transcripts by a C-terminal extension bacteria), purine phosphoribosyltransferase (in archaea)
containing 4 tandem copies of the wHTH domain [44]. and the threonine synthase (restricted to the genus Pyro-
We also detected a wHTH domain, similar to those in coccus) (Fig. 3). Some of these architectures, like BirA
SelB at the N-terminus of the bacterial RNA-processing are widely distributed in the prokaryotic genomes and
enzyme Rnase R, which might bind RNA (Fig. 3). How- appear to be ancient. Others like the fusion of an OmpR
ever, in proteobacteria the Rnase R is also known to family wHTH with the uroporphyrinogen-III synthase
function as regulator of virulence genes [79], suggesting are found only in actinobacteria, while yet others like
that this HTH domain also additionally functions as a a fusion to the threonine synthase are restricted to a sin-
conventional transcription regulator. gle genus, and are apparently of more recent prove-
 L. Aravind et al. / FEMS Microbiology Reviews 29 (2005) 231–262 243

nance. This observation suggests that the combinations 3.4. Architectures related to two-component, PTS and
of HTHs with enzymatic domains have been repeatedly serine/threonine kinase signaling
selected for throughout the span of prokaryotic
evolution. The two component phospho-relay system, involving
Two other specialized classes of domain architectures the histidine kinase and the receiver domain, which is
arise through fusions of the HTH domains with either of phosphorylated on a conserved aspartate, comprise
two types of P-loop NTPase domains, namely the NtrC- one of the most common signaling systems in bacteria
like AAA+ domains [84] and the STAND (signal trans- and certain archaea [90,91]. The fusions of the receiver
duction ATPases with numerous domains) NTPase domain with the HTH are typical of transcriptional reg-
domain [85]. Proteins containing the NtrC-like AAA+ ulators responding to histidine kinase-dependent signal-
domains are found only in those bacteria that contain ing. Two of the most common architectures, seen in the
sigma-54, and they bind a distant enhancer element majority of bacteria, involve combinations of a single N-
and activate transcription of sigma-54 bound promot- terminal receiver domain to either a LuxR-like tetra-
ers. The AAA+ ATPase domains of these proteins per- helical HTH domain (e.g. UhpA and NarL) or wHTH
form an ATP-dependent chaperone-like activity that domain (e.g. OmpR and PhoB, Fig. 4). Less frequent fu-
converts the ‘‘closed’’ sigma-54-containing transcription sions involving HTH domains of the AraC- and the
complexes to an ‘‘open’’ configuration, which is favor- CitB families are seen in certain bacteria. Other than
able for transcription initiation [84]. The NtrC-like these simple architectures, several more complicated
AAA+ domains are fused to at least two different types architectures involving multiple receiver domains or
of HTH domains. The classical versions like NtrC and even fusions to additional histidine kinase (e.g. B. cereus
TyrR are fused to a C-terminal basic tri-helical HTH protein BC3207) and NtrC-like AAA+ ATPase (e.g. E.
domain of the Fis family [86]. The second version typi- coli NtrC) domains are also observed (Fig. 3) [90,91].
fied by the Bacillus levanase operon regulator, LevR, in- The PTS sugar-transport systems [92] use a phospho-
stead contains an N-terminal wHTH domain, suggesting relay cascade to transfer a phosphate from phosphoenol
that there have been two independent fusions of the pyruvate to a histidine on the PTS regulatory domain
HTH domain with NtrC-like AAA+ ATPases (Fig. 3). (PRD), which often co-occurs in the same polypeptide
The STAND P-loop NTPases are, as a rule, large mul- with HTH domains [93,94]. The PRDs receive the phos-
ti-domain proteins that appear to catalyze the ATP- phates from the HPr and EIIB proteins of the PTS sys-
dependent assembly of complexes in variety of signaling tem, and depending on their phosphorylation state
contexts [85]. They typically contain repetitive super- regulate transcription. Architectures involving the
structure-forming domains, such as the WD and TPR PRD domain are analogous to those involving the recei-
domains, which may serve as surfaces for the assembly ver domain of the two-component system. The simplest
of multi-protein complexes [85]. The archetypal mem- versions contain an N-terminal wHTH domain fused to
bers of the architectural class combining the HTH and a C-terminal PRD domain. The more complex forms
STAND domains are the E. coli MalT [87], Bacillus contain more than one PRD domains, or fusions to
GutR [88] and Streptomyces AfsR proteins [89]. A re- NtrC-like AAA+ domains and PTS system EIIB do-
cent analysis of the STAND superfamily revealed that mains, which determine sugar specificity [95,96] (Fig.
the HTH domains have been fused to them on several 3). The B. subtilis LicR protein contains an N-terminal
independent occasions [85]. The fusions involving the HTH fused to two PRDs and both EIIB and EIIA com-
OmpR family of wHTH domains (e.g. in AfsR) usually ponents of the PTS system [95,96], indicating that it is a
link the HTH to the N-terminus of the STAND NTPase multi-functional protein that directly regulates both su-
domain. In contrast, fusions involving the LuxR family gar uptake and transcription of sugar-utilization genes.
of HTH link it to the C-terminus of the STAND mod- The 3H domain, which is related to the HPr domain
ule, with a set of a-helical repeats occurring between of the PTS system, is also found fused to BirA-related
these two modules (e.g. GutR and MalT) (Fig. 3). The wHTH domain in several bacterial proteins typified by
STAND-domain-containing transcription regulators Tm1602 from Thermotoga maritima [97]. The 3H do-
are likely to integrate multiple signaling inputs via inter- main may represent another novel domain that may be
actions of their STAND and super-structure forming regulated by phosphorylation on its conserved histi-
domains, and are particularly prevalent in the develop- dines, perhaps via a PTS-like system.
mentally or organizationally more complex bacteria. The serine–threonine kinases are over-represented in
Another version of the HTH domain, associated with certain organizationally complex bacteria, like the cya-
a restriction endonuclease fold and STAND NTPases nobacteria and the actinobacteria. In the latter group
domains (Fig. 3), is found in the PH-type ATPases that there is class of proteins, typified by the protein EmbR,
are expanded in Pyrococci. These domains have been containing a fusion of the HTH domain with the FHA
predicted to localize the endonuclease domains of these domain [98]. The FHA domain in this protein binds
proteins to their target sequences [30]. phosphoserine peptides, and mediates its interaction
244 L. Aravind et al. / FEMS Microbiology Reviews 29 (2005) 231–262

Fig. 4. Phyletic patterns and demography of selected families of HTH domains in selected prokaryotic proteomes. The bar graph depicts actual
counts of the number of HTHs in each family per genome. Species abbreviations are as follows: Ap: Aeropyrum pernix, Atu_w:Agrobacterium
tumefaciens C58 (U. Washington), Aae: Aquifex aeolicus, Af: Archaeoglobus fulgidus DSM 4304, Bs: Bacillus subtilis, Bth_V:Bacteroides
thetaiotaomicron VPI-5482, Blo: Bifidobacterium longum, Bbr: Bordetella bronchiseptica, Brja: Bradyrhizobium japonicum, Ccr: Caulobacter
crescentus, Ct: Chlamydia trachomatis, Ctep: Chlorobium tepidum, Ctet: Clostridium tetani E88, Cgl: Corynebacterium glutamicum, Dr: Deinococcus
radiodurans, Tth: Thermus thermophilus, Dvu: Desulfovibrio vulgaris, Ec_C:Escherichia coli CFT073, Fnu: Fusobacterium nucleatum, Gsu: Geobacter
sulfurreducens, Gvi: Gloeobacter violaceus, Hasp: Halobacterium sp. NRC-1, Lint: Leptospira interrogans serovar lai 56601, Mlo: Mesorhizobium loti,
Mj: Methanococcus jannaschii, Mac: Methanosarcina acetivorans, Mth: Methanothermobacter thermautotrophicus, Mtu_H:Mycobacterium tubercu-
losis H37Rv, Neq: Nanoarchaeum equitans, Neu: Nitrosomonas europaea, Ana: Anabaena sp. PCC 7120, Oyp: Onion yellows phytoplasma, Pto:
Picrophilus torridus, Psp: Pirellula sp., Pgi: Porphyromonas gingivalis, Pae: Pseudomonas aeruginosa, Pyae: Pyrobaculum aerophilum, Pa: Pyrococcus
abyssi, Rpa: Rhodopseudomonas palustris, Son: Shewanella oneidensis, Sme: Sinorhizobium meliloti, Sau_MR:Staphylococcus aureus subsp. aureus
MRSA252, Sav_MA:Streptomyces avermitilis MA-4680, Scoe: Streptomyces coelicolor, Sso: Sulfolobus solfataricus, Sth: Symbiobacterium
thermophilum, Ssp: Synechocystis sp. PCC 6803, Tm: Thermotoga maritima, Vch: Vibrio cholerae, Wsu: Wolinella succinogenes.

with the upstream protein kinase in regulating the bio- 3 and 4). In their simplest form they combine a HTH do-
genesis of the mycobacterial cell wall [99]. Taken to- main with a small molecule binding domain (SMBDs)
gether, HTH domains fused to the receiver, PRD, 3H [97]. More complex architectures may involve multiple
and FHA domains represent a distinct class of architec- SMBDs or even additional domains such as the NtrC-
tures that are typical of proteins responding to environ- like AAA+. The same SMBDs found in the single com-
mental and physiological stimuli downstream of ponent systems may also occasionally be found fused to
signaling cascades. two-component regulators, where they may supply sec-
ondary allosteric inputs (Fig. 3).
3.5. Architectures related to single-component signaling The most common SMBDs fused to HTHs in the sin-
gle component systems are drawn from a relative small
In contrast to the above-discussed signaling cascades, set of ancient protein folds (Table 1): (1) The PAS-like
the single-component systems are defined as those sig- fold, with representatives such as the PAS domain, the
naling systems in which the transcription regulatory do- GAF domain, and the ligand binding domains of the
main and the stimulus sensor domain are combined in a IclR-type transcription factors [66,100]. (2) The periplas-
single protein. These architectures, which are function- mic binding protein types I and II domains, which in-
ally analogous to the fusions of the HTHs with the met- clude the ligand-binding domains of the LysR family
abolic enzymes, are by far the most prevalent [101–103]. (3) The ferredoxin-like fold, which includes
architectural category in prokaryotes (Table 1 and Figs. the ACT domain and related ligand-sensing domains
 L. Aravind et al. / FEMS Microbiology Reviews 29 (2005) 231–262 245

of the Lrp-like transcription factors and the classic ferre- sensor for the assembly state of certain multi-protein
doxins, which are fused to HTH domains in archaeal complexes. Hence, transcription factors like IagA with
and cyanobacterial proteins [104–106]. (4) The double- the TolB-N-related domains might regulate transcrip-
stranded b-helix (cupin) fold, which contains the tion in response to the dynamics of multi-protein
AraC-type ligand-binding domains, as well as the cNMP complexes.
binding domains [97,107]. (5) The CBS domain that oc-
curs as an obligate dyad [108]. (6) The GyrI domain, 3.6. Unusual functional adaptations of the HTH domain
which contains two copies of the SHS2 structural mod-
ule, appears to be one of the principal ligand-binding Beyond its usual DNA binding role the HTH domain
domains of the MerR family [109]. appears to have been exapted for a variety of functions,
Some other SMBDs share a common fold with enzy- where it is utilized as a molecular adaptor. For example
matic domains, but appear to be catalytically inactive the permuted version of the wHTH in the N-termini of
versions that merely bind low-molecular weight sub- the methionine aminopeptidases appears to represent an
strates. Examples of these are: (1) the UTRA domain, ancient recruitment to a protein–protein interaction
which is found in the HutC/FarR group of GntR family function [60]. Several such instances of recruitment of
transcription factors and possesses the same fold as the HTH domain to protein–protein interactions are
chorismate lyase [110] and (2) The DeoR ligand-binding seen in the eukaryotes. One such example is the PINT
domain, which shares a common a/b fold, which is also domain, which forms the structural scaffold of the pro-
present in the enzymes of the phosphosugar isomerase teasomal lid, the signalosome and the eukaryotic initia-
family such as ribose phosphate isomerase [111]. The tion factor eIF3 [117,118]. It appears to have been
enormous genomic information has resulted in the avail- derived from a prokaryotic wHTH precursor which sec-
ability of proteins from numerous prokaryotes, display- ondarily lost its DNA-binding properties. The Snf8 fam-
ing several new domain architectures. In many of these ily of proteins in eukaryotes contains two tandem copies
proteins, uncharacterized globular domains are fused of a wHTH domain related to the PINT domain. This
with the HTH and other signaling domains, in architec- family includes the Vps22, Vps25 and Vps36 proteins,
tures analogous to those of known sensory domains. which are required for sorting of transmembrane pro-
Thus, these analogous architectures enable the predic- teins and lipids into the multivesicular-bodies in the
tion of novel sensory domains of one-component sys- eukaryotic vesicular transport system [119–121]. Just
tems. By this procedure several new candidate sensory as in the case of the PINT domain, the duplicate wHTH
domains, with somewhat lower abundance than the pre- domains of the Snf8 family proteins provide a scaffold
viously described domains, were uncovered (Table 1). for the formation of multi-protein ESCRT complexes
An example of such a domain is suggested by the PocR required for vesicular trafficking. Additionally, the same
protein, from Salmonella, with an AraC-like HTH do- complex of Snf8 family proteins are also implicated in
main, which binds the effector 1,2-propanediol, and reg- transcriptional elongation [119], suggesting that they
ulates the propanediol regulon [112]. It contains a might have been secondarily recruited for a eukaryote-
distinct globular N-terminal domain that is also found specific role in vesicular transport from an original role
fused to histidine kinases, chemotaxis receptors, and in transcriptional regulation. The cyclins and Retino-
diguanylate phosphodiesterases of the HD-GYP family blastoma are derivatives of the ancestral TFIIB protein
in other bacterial proteins (VA and LA unpublished). which were utilized for specific protein–protein interac-
These domain combinations suggest that it a novel evo- tions, respectively involved in regulating the eukaryotic
lutionarily mobile, small-molecule-sensing domain, cell-cycle controlling kinases and the E2F/DP1 proteins
which probably initiates responses through the domains [122]. Likewise, the DEP domain, which is found in sev-
with which it is linked. eral signaling proteins [49], the cullin C-terminal domain
We also found a conserved domain in the Salmonella (found in cullins, which are the adaptors for the ana-
typhi invasion regulator IagA [113], which occurs C-ter- phase-promoting ubiquitin ligases) [50], and the C-ter-
minal to the OmpR-like wHTH domain domain (Fig. 3). minal domain of Esa1-like histone acetylases [123] are
Additionally, it occurs independently in other bacterial other notable examples in which the wHTH domain
proteins fused to adenylyl cyclase and histidine kinase has been recruited to mediate specific protein–protein
domains (data not shown). Iterative sequence profile interactions in diverse eukaryote-specific signaling con-
searches suggest that this domain shares a common fold texts. In most of these proteins the HTH domain sticks
with the TolB N-terminal domain [114,115], and is typ- out as a distinct domain in a complex modular architec-
ically found at the N-termini of super-structure forming tural context.
repeats such as TPR and WD40 repeats (Fig. 3). These The plant isoflavone O-methyltransferases contain a
architectures, and the interactions of the TolB protein N-terminal wHTH domain which is related to that in
[116], suggest that this domain probably acts in unison bacterial transcription factors, and appears to function
with the super-structure forming proteins as a potential as a dimerization domain [124]. The closest relatives of
246 L. Aravind et al. / FEMS Microbiology Reviews 29 (2005) 231–262

these plant methyltransferases are seen in bacteria (e.g. and its derivatives. Beyond these, there are few other
McmR from Streptomyces lavendulae) suggesting that highly derived versions whose provenance is hard to
the plant lineage probably acquired these enzymes establish on account of their extreme sequence and
through lateral transfer from a bacterial source. Hence, structure divergence. Below, we briefly describe the ma-
it is possible that the wHTH domain in the bacterial pre- jor evolutionary lineages of the HTH along with their
cursor originally functioned as a transcription regulator phyletic patterns and functional diversification.
that was fused to the methyltransferase domain (see
above for discussion on analogous fusions) but was sub- 4.1. Lineages of basic tri-helical HTH domains
sequently reused in the plants in a structural role. A sim-
ilar case is presented by the carbamoylphosphate Several distinct families that retain the primitive sim-
synthetase (CPS), which contains a tandem duplication ple tri-helical HTH domain are represented in one or
of HTH domains between the carboxyphosphate and more of the major divisions of life. The duplicate
carbamoyl phosphate synthetic modules of the enzyme HTH domains found in the carbamoyl phosphate synthe-
(Fig. 3). These HTH domains are of the simple tri- tase represent a distinctive lineage of simple trihelical
helical versions, like those encountered in bacterial tran- domains present in all the 3 super-kingdoms of life.
scription factors such as Fis and LuxR. However, in Phylogenetic trees show that these proteins follow the
CPS, rather than binding DNA, they apparently func- ‘‘standard model topology’’, with a distinct archaeo-
tion as protein–protein interaction domains that convert eukaryotic branch and a bacterial branch [126]. This
the enzyme to its oligomeric form in the presence of topology and their phyletic pattern suggest that this line-
uridine [125]. age most probably, goes back to the LUCA. The HTH
domains bearing the catalytic tyrosine of the topoiso-
merase I family that is found in all the 3 super-kingdoms
4. The evolutionary classification of HTH domains and the archaeal topoisomerase VI are also distinctive
lineages of tri-helical HTH domains with no specific
As the HTH is a small domain, which exhibits ex- relationship to any other HTH domains. The phyletic
treme sequence divergence, reconstruction of its higher- pattern of the former lineage suggests that it was prob-
order natural classification is fraught with problems ably present in the common ancestor of the 3 super-
arising from the erosion of evolutionary signal. Never- kingdoms [127].
theless, the availability of numerous high-resolution Rbp10 family, which is defined by the eponymous
structures and extensive sequence information allows RNA polymerase core subunit [54,128], is universal in
us at least to reconstruct the major evolutionary radia- both archaea and eukaryotes and appears to have been
tions of this domain. This reconstruction is based on part of the shared vertical inheritance of the archaeo-
three distinct sources of information: (1) Structural fea- eukaryotic lineage. Likewise the sigma factor family
tures (see above for discussion) help in establishing the [129] is conserved throughout the bacteria but bona fide
relationships at the highest level. (2) Sequence informa- representatives of this group are absent in the archaea
tion can be used for clustering based on similarity and eukaryotes. Members of this group are character-
scores, conventional phylogenetic analysis and cladistic ized by a tandem duplication of the HTH domain. sig-
analysis with discrete sequence characters. These se- ma-70, which is the basal transcription factor of the
quence-based procedures help in resolving the relation- bacteria, is usually present in single copy in all bacterial
ships at a lower level, such as defining the principal lineages and shows a noticeable phylogenetic signal sug-
sequence families, the relationships within them, and gestive of a largely vertical inheritance since the last
some of the higher-level groupings between sequence common ancestor of all the extant bacteria [129,130].
families. (3) Phyletic patterns (Fig. 4) help in recon- In contrast other sigma factor subfamilies show evidence
structing the temporal aspects of the evolutionary his- for lateral transfer, gene loss and lineage specific expan-
tory of these domains and also help in constraining sions (Fig. 4). In particular, the ECF subfamily appears
the directions of derivation of particular versions from to have been widely expanded in numerous bacterial lin-
others. The combined scenario gleaned from these direc- eages, especially in those with complex metabolic and
tions is presented schematically in Fig. 5 (for details of developmental capabilities [129]. The lineage specific
the combined approach see [85]). diversification of the ECF subfamily of sigma appears
This higher-order evolutionary scheme is character- to have been a major contributor to the evolution of
ized by the presence of several basal lineages that retain niche-specific adaptations in bacteria by allowing di-
the primitive basic tri-helical version HTH fold, but verse patterns of differential gene expression (see below).
have no other shared-derived character that groups The sigma-54 family appears to have been derived inde-
them together. These basal lineages are followed by pendently from the remaining sigma factors and is an-
the two great monophyletic lineages, namely the tetra- other distinct lineage of tri-helical HTHs that is
helical superclass and its derivatives and the wHTH sporadically distributed in bacteria.
 L. Aravind et al. / FEMS Microbiology Reviews 29 (2005) 231–262 247

Fig. 5. Higher order evolutionary relationships of HTH domains. The horizontal lines show temporal epochs corresponding to three major
transitions in evolution, the last Universal common ancestor, the diversification of archaea and bacteria and the evolution of the eukaryotes. Solid
lines reflect the maximum depth of time to which a particular family can be traced. Broken lines indicate an uncertainty with respect to the exact
point of origin of a lineage. Colored circles at the termini of the lines represent broad functional classes: where yellow represents DNA binding, pink
represents RNA binding and blue, interaction with proteins. The ellipses encompass groups of lineages from which a new lineage with relatively
limited distribution could have potentially emerged. Lineages of archaeal origin are colored blue, those of bacterial origin are colored orange and
those present in archaea and bacteria are colored black. Lineages only detected in the eukaryotes are colored green. The yellow triangle reflects the
origin of the L11 family of proteins, the blue triangle reflects the origin of the winged HTHs and the red triangle reflects the origin of the tetra-helical
version. The phyletic distribution of the lineages are also shown in brackets, where A: Archaea; B: bacteria, E: eukaryotes, proteo: Proteobacteria
and Crown: Crown group eukaryotes. The Ô>Õ reflects lateral transfer with the arrow head pointing to the potential direction of transfer.
248 L. Aravind et al. / FEMS Microbiology Reviews 29 (2005) 231–262

The Fis family of basic tri-helical HTH domains is lating residues. The KorB–ParB family also contains a
also found solely in the bacteria and typically appears basic tri-helical version of the HTH domain, and func-
at the C-termini of the NtrC-like AAA+ domains tions as the partitioning protein for diverse bacterial
[131]. It appears to have emerged early in bacterial evo- plasmids. The KorB subfamily contains an additional
lution and spread widely via lateral gene transfer along C-terminal 4-helical DNA-binding domain [57] fused
with the spread of sigma factors of the sigma-54 family. to the HTH domain, while the ParB subfamily contains
The Fis protein itself appears to have been secondarily a fusion to a nuclease domain of the OB-fold [139].
derived in the proteobacteria through the gene fission The MetJ-Arc (RHH) family of transcription factors
of an NtrC-like regulator [131]. Likewise, the trihelical appears to have been derived from the basic tri-helical
HTH domains of the Rok family are restricted to the bundle [61]. They are most frequently found as tran-
bacteria and always found in combination with sugar ki- scriptional regulators of the mobile toxin–antitoxin
nase domains of Hsp70/actin fold. The archaeal Boa1 operons [140]. Hence, it is possible that they were origi-
family of tri-helical HTH domains contains a unique nally derived in such toxin–antitoxin systems, through
all b-strand domain at the N-terminus that is likely to rapid divergence from a conventional HTH. This ap-
bind its effectors (Table 1, Fig. 4). The Myb family is a pears to have happened early in the evolution of one
pan-eukaryotic family of simple tri-helical domains that of the prokaryotic lineages, after which they were widely
appears to have diversified into multiple members prior disseminated across the prokaryotes through horizontal
to the diversification of all extant eukaryotic lineages mobility.
(Fig. 5). Some versions of the Myb family, the SANT
subfamily, appear to have been recruited secondarily 4.2. The tetra-helical HTH superclass and its derivatives
for protein–protein interactions in the eukaryotic chro-
matin [132]. In bacteria, the Myb domain is only seen The first major monophyletic clade of HTH domains
in the RsfA-related pre-spore transcription factors of is defined by the unifying structural feature of the tetra-
the low-GC Gram-positive bacteria [133], suggesting helical bundle. Sequence similarities help in identifying
that it was acquired relatively late through lateral trans- several major lineages within this group. The cI-like
fer from the eukaryotes. In addition to the Myb domain, family, typified by the phage lambda cI protein, contains
the homeodomain and POU domain families [41], which representatives from across the 3 super-kingdoms of life
are also basic tri-helical HTH domain, are respectively (Fig. 4). Several distinct subfamilies can be recognized
widespread in the crown-group eukaryotes and metazo- within this family. The largest of these is the bacterial
ans. The HTH domain of the eukaryotic tumor suppres- repressor subfamily typified by the protein PbsX (Xre)
sor BRCA2 [72] is another simple trihelical version that from the B. subtilis prophage 168 [141]. Another notable
was derived early in eukaryotes. However, it shows no subfamily is the MBF1 subfamily, which is nearly uni-
specific relationships to any other HTH domains with versally conserved in the archaeo-eukaryotic lineage
a similar structural configuration. functions, and is an adaptor that appears to bridge the
Beyond these classical families there are several tri- specific transcriptional regulators to the basal transcrip-
helical HTH domains associated with diverse transpos- tion machinery MBF1 [142].
ases and resolvases, such as the gamma–delta resolvase. The next major assemblage within the tetra-helical
The exact point of origin of the HTH domains associ- superclass comprises of 6 major families that are exclu-
ated with these mobile elements is difficult to ascertain, sively prokaryotic in their distribution (Figs. 4 and 5).
but they appear to have given rise to families of HTH This assemblage includes the AraC, LuxR, LacI, DnaA,
domains found in cellular transcription factors on multi- TrpR and TetR families, which are predominantly bacte-
ple occasions. Particularly striking examples of these in- rial with several independent lateral transfers to archaea
clude the Paired box and Pipsqueak families involved in (Fig. 4). The first four of these families are nearly pan-
metazoan developmental gene expression, and the bacterial in their distribution suggesting that these
CENBP family (centromere binding protein) in the HTH families had probably diverged from each other
crown group eukaryotes [134–137]. Likewise, the HTH even in the common ancestor of all bacteria (Fig. 5).
domain of the bacterial YlxL(SwrB) family (Fig. 3) is The latter two lineages are more limited in their distribu-
also related to the HTH domains of the gamma–delta tion, but are found in most proteobacteria, and low GC
resolvase [138]. It is possible that other transcription Gram positive bacteria (Fig. 4). Within some of these
factors families with a relatively restricted phyletic dis- families several distinct lineages, often defined by spe-
tribution, like the eukaryotic homeodomain, and the cific architectural themes can be identified. However,
bacterial sigma-54 family have also ultimately been de- most of these families contain a dominant architectural
rived from the HTH domains of transposases. The me- theme suggesting these might have been the earliest ver-
tal-binding domain of the retroviral integrase also sions of these families. The AraC family contains a
appears to have been derived from the HTHs of trans- duplication of the tetra-helical version of the HTH do-
posase/resolvase class through acquisition of metal-che- main [143], and typically occurs fused to a sugar binding
 L. Aravind et al. / FEMS Microbiology Reviews 29 (2005) 231–262 249

domain of the DSBH fold [97,107] suggesting that they these families, barring some striking exceptions (see be-
predominantly function as sugar-sensing transcription low) function as transcription factors suggesting that
factors. However, in complex cyanobacteria, like Nos- they could have descended from an ancestral protein
toc, the majority of AraC family HTH domains occur that had some role in transcriptional regulation. The
fused to a novel sensor domain that is also found in MarR family has vastly proliferated in the archaea to
the siderophore alcalgin sensing transcriptional activa- give rise to several archaeal subfamilies and includes
tor, AlcR, from Bordetella [144] (Fig. 6 and Table 1). most of the major archaea-specific wHTH transcription
The most common subfamily of the LuxR family con- factors. In bacteria the RuvB sub-family, which is de-
tains fusions to the receiver domain, as seen in the case rived from the ancestral HrcA lineage, appears to have
of the E. coli protein NarL. In the case of the LacI fam- secondarily acquired a role in DNA recombination after
ily the dominant architecture features a fusion of the a fusion with the AAA+ domain in the ATPase subunit
HTH domain with a small-molecule binding domain of the Holliday junction resolvase [148]. Another sub-
of the periplasmic solute-binding protein fold. DnaA is family of the HrcA–RuvB family, S19AE is a small sub-
usually found only in a single copy in all bacterial gen- unit ribosomal protein in the archaeo-eukaryotic
omes, with the HTH occuring at the C-terminus of the lineage, with a potential RNA-binding function (Fig.
AAA+ domain [145]. The DnaA protein functions in 5). A similar case is observed in the GntR family, which
replication initiation, and also as a transcription factor has vastly proliferated in bacteria giving rise to many of
[146]. Additionally, sporadic versions of the tetrahelical the major bacterial one-component transcription fac-
HTH superclass are also seen in several phage transpos- tors. However, in the archaeo-eukaryotic clade it is rep-
ases related to the Mu transposase. resented by a single lineage, the small subunit ribosomal
TFIIB, a basal transcription factor in the archaeo- protein S25AE (Figs. 4 and 5).
eukaryotic lineage, defines the TFIIB family, a derivative In contrast to the above families, the BirA, ModE,
of the tetra-helical class. In the archaea there is typically PadR, ScpB and DtxR-Fur families appear to have
a single version of this family (Fig. 4). However, in the had a bacterial origin followed by sporadic lateral trans-
eukaryotes not only did TFIIB undergo duplication, fers to the archaea (Fig. 4). The DtxR–Fur family
but it also spawned two more divergent families, namely appears to have specialized early on in regulating me-
the cyclins and the Rb proteins [122]. Structural com- tal-dependent transcription of genes. The ScpB family
parisons suggest that the eukaryote-specific Bright do- is typically represented in most bacteria by a single pro-
main family [55,147], which includes DNA-binding tein, which is encoded in the same operon as a kleisin
proteins involved in chromatin dynamics was also de- and a SMC-type ABC ATPase [149,150]. The ScpB pro-
rived from a TFIIB-like precursor prior to the radiation tein contains a tandem duplication of the wHTH with a
of eukaryotes from their common ancestor. The prove- C-terminal helix after the wing (Fig. 3), and has been
nance of the only bacterial relatives of this version, shown to regulate the activity of the chromosome re-
namely the Spo0A protein from endospore-forming bac- organizing SMC ATPases [149,150]. The archaeal repre-
teria [56] remains unclear. sentatives of this family appear to have been acquired
through a lateral transfer from the cyanobacteria
4.3. The wHTH superclass (LMI and LA unpublished). The pan-bacterial families,
namely CitB, LysR and Rrf2, are largely absent in the
The second major monophyletic clade of HTH do- archaea (Fig. 4). Interestingly, a single member of the
mains are unified by the presence of a striking derived Rrf2 family (GLP_14_27362_29578) is seen in the early
feature, the ‘‘wing’’. Several major assemblages with branching eukaryote Giardia lamblia. A number of
varying distributions can be identified within the wHTH wHTH domains with distinct sequence conservation
superclass. The wHTH superclass includes the majority profiles, and occurring in large multi-domain proteins
of prokaryotic transcription factors. Thirteen major also belong to this assemblage of wHTH domains with
families of prokaryotic wHTH domains, namely the a C-terminal helix after the wing. These include (1)
BirA, ArsR, GntR, DtxR-FurR, CitB, LysR, ModE, two HTH domains of the topoisomerase II family, (2)
MarR, PadR, YtcD, Rrf2, ScpB and HrcA-RuvB fami- the HTH domain of the enigmatic topoisomerase V,
lies, are unified by the presence of a characteristic helix which is currently found only in Methanopyrus kandleri
after the wing, and comprise the largest monophyletic [78], (3) The ESA1 family of eukaryotic histone acety-
assemblage within the wHTH superclass (Fig. 5). Of ltransferases and (4) the N-terminal HTH domains of
these, representatives of the ArsR, MarR, YctD, GntR, the Rio kinases from archaea and eukaryotes. Two re-
HrcA-RuvB are seen in both archaea and bacteria (Fig. lated wHTH domains from Giardia (Genbank Gis:
4) with phylogenetic trees suggesting distinct pan-archa- 29245940, 29247865) also appear to have been derived
eal and pan-bacterial branches within them (data not from within the above-discussed assemblage of wHTH
shown). This would imply that these families possibly domains, but their precise affinities are obscured due
even go back to the LUCA (Fig. 5). The members of to rapid sequence divergence.
250 L. Aravind et al. / FEMS Microbiology Reviews 29 (2005) 231–262

Fig. 6. A multiple alignment of the AlcR N-terminal module. Multiple sequence alignment the AlcrN domain was constructed using T-Coffee after
parsing high-scoring pairs from PSI-BLAST search results. The Jpred secondary structure is shown above the alignment with H representing an a-
helix and E representing a b-strand. The 85% consensus shown below the alignment was derived using the following amino acid classes: hydrophobic
(h: ALICVMYFW, yellow shading); small (s: ACDGNPSTV, green) and polar (p: CDEHKNQRST, blue). The conserved ÔCÕ is shaded red. The
limits of the domains are indicated by the residue positions, on each end of the sequence. The sequences are denoted by their gene name followed by
the species abbreviation and GenBank Identifier (gi). The species abbreviations are: Ana: Nostoc sp., Bbro: Bordetella bronchiseptica, Bpar:
Bordetella parapertussis, Ctet: Clostridium tetani, Ecar: Erwinia carotovora, Gvio: Gloeobacter violaceus, Pae: Pseudomonas aeruginosa, Plum:
Photorhabdus luminescens, Psyr: Pseudomonas syringae, Rpal: Rhodopseudomonas palustris, Rsol: Ralstonia solanacearum, Smel: Sinorhizobium
meliloti, Spne: Streptococcus pneumoniae, Ssp: Synechocystis sp., Styp: Salmonella typhimurium, Tden: Treponema denticola, Wsuc: Wolinella
succinogenes.
 L. Aravind et al. / FEMS Microbiology Reviews 29 (2005) 231–262 251

The next major monophyletic assemblage of wHTH protein S10 and the bacterial selenocysteine-specific
superclass includes the DeoR, ArgR, LevR, YitL, Lrp- elongation factor SelB appear to comprise a family prin-
AsnC, ZBD (Z-DNA binding domain), and RNase R cipally associated with translation and RNA metabo-
families. These families are unified by overall sequence lism proteins (Figs. 3 and 5). Their phyletic patterns
similarity, and a conserved pattern with a conserved glu- suggest that their recruitment to RNA-specific functions
tamine or arginine residue between helix-1 and helix-2 of appears to have occurred after the separation of the ma-
the HTH domain. Of these the Lrp-AsnC family is jor superkingdoms of life, though it is possible that a
widely conserved in both bacteria and archaea (Figs. 4 standalone precursor of this family was already present
and 5) and in phylogenetic trees displays distinct in the LUCA. Another distinct family of wHTH do-
branches separating the majority of archaeal and bacte- mains with an exclusive single-stranded RNA-binding
rial members. Hence, it is possible that the Lrp-AsnC function is the La domain family [47,48,157]. The La do-
family goes back to the LUCA. The ArgR and DeoR main has previously only been reported from eukary-
are predominantly bacterial families, whereas the LevR otes; however, using sequence profile analysis we show
group is sporadically found, mainly in low GC Gram that it is homologous to the N-terminal domain of the
positive bacteria. The RNase R family is a limited group NAD-dependent RNA 2 0 -phosphotransferase [158],
that is represented by just a single pan-bacterial orthol- which removes the phosphate from the 2 0 ends of
ogous lineage in the form of the wHTH domain at the RNA. In the RNA 2 0 -phosphotransferase the La do-
extreme N-terminus of the Rnase R protein (Fig. 3). main bears two of the four absolutely conserved cata-
Its widespread distribution in the bacteria suggests that lytic histidines (LA unpublished), suggesting that it is
it emerged early in the evolution of this lineage. The another case of recruitment of the HTH domain for a
ZBD family is restricted to the crown group eukaryotes catalytic role. The RNA 2 0 -phosphotransferases are
and in animals it is fused to the deaminase domain in- highly conserved in the archaeo-eukaryotic and sporad-
volved in hyper-mutation of the immunoglobulin genes ically observed in the bacteria. This suggests that the La
[151,152]. The restricted phyletic pattern of the ZBD family of HTH domains emerged early in the archaeo-
family suggests that it may have evolved from one of eukaryotic lineage and were subsequently laterally trans-
the prokaryotic families after lateral transfer to the ferred to bacteria. In the eukaryotes the non-catalytic
crown group eukaryotes (Fig. 5). versions (the classical La domains) were recruited for
The wHTH domains found in the archaeo-eukaryotic binding 3 0 poly(U)-rich elements of nascent RNA poly-
proteins involved in replication initiation, namely the merase III transcripts and translation regulation [47,48].
MCM proteins, the CDC6–Orc1 proteins (C-terminal The fungal protein frequency [157] contains an as yet
to the AAA+ ATPase domains in both these cases) functionally uncharacterized version of the La domain,
and the RP-A protein comprise the replication initiation which may regulate the circadian clock via RNA
family of wHTH domains. Both the MCM and the metabolism.
CDC6 versions appear to have been present right from There are other distinct families of wHTH transcrip-
the base of the archaeo-eukaryotic lineage [30,153,154]. tion factors in prokaryotes with related 2- or 3- stranded
The version associated with RP-A occurs as a stand- wHTH domains, but they do not appear to belong to
alone protein in the archaea, while it appears to have any of the aforementioned assemblages. These include
been fused to the single strand DNA binding OB fold the LexA, OmpR, and IclR families that appear to be
domains in the eukaryotes. Also belonging to this family pan-bacterial families, with at best a rare presence in
are the C-terminal wHTH domains from the replicative the archaea (Figs. 4 and 5). The classical representatives
helicase-primase enzymes of various viruses such as P4, of the LexA family appear to be involved in regulating
plasmid Rep proteins and the eukaryotic RFX-type responses to DNA damage in diverse bacteria [82].
DNA binding domain seen in transcription factors like However, a highly divergent, potential offshoot of the
the MHC class II transcription factor HRFX1 LexA family is seen fused to the C-termini of large
[75,155,156]. This family might have originally evolved DNA helicases (lhr) that are found sporadically in sev-
to recognize specific DNA features associated with the eral bacteria (Fig. 3). The eukaryotes display their own
replication initiation sites, and recruiting the catalytic families of specific transcription factors, such as the
activities involved in pre-initiation and initiation to Forkhead-histone H1 [25,26] and E2F-DP1 families
these sites [75]. The RFX domains are specifically re- [159], and the basal transcription factors, such as the
lated to certain phage helicase-primase wHTH domains TFIIF(Rap30)-TFIIIC-63K family [160] that show
belonging to this family. This suggests that the crown structurally similar wHTH domains, but lack specific se-
group eukaryotes may have acquired the RFX domains quence relationships with any of the prokaryotic fami-
from such a viral replication protein and reused them as lies. Within the TFIIF-TFIIIC-63K family, the RNA
a transcription regulator (Fig. 5). polymerase III transcription factor, TFIIIC-63K, is con-
The wHTH domains of the archaeal phenylalanyl served throughout eukaryotes, but TFIIF appears to be
tRNA synthetase a-subunits, the eukaryotic ribosomal restricted to the crown group and the apicomplexans.
252 L. Aravind et al. / FEMS Microbiology Reviews 29 (2005) 231–262

The wHTH domains of the TFIIF and TFIIIC-63K are distribution. A potential RNA-binding version of this
functionally similar to those of the Forkhead-histone H1 domain is the N-terminal domain of translation initia-
family suggesting that the latter were probably derived tion factor IF2 from certain bacteria [165]. The remain-
from more widespread family of basal transcription fac- ing versions, observed in the phage lambda excisionase
tors [160]. The remaining eukaryotic families appear to and terminase proteins, the phage Mu-repressor family
be chiefly represented in the eukaryotic crown group, and the eukaryotic DNA repair protein XP-A and ani-
implying that they arose relatively late from pre-existing mal transcription factors of the Dachshund family, ap-
eukaryotic wHTH domains found in the basal transcrip- pear to be DNA binding [166–168]. Eukaryotic Xp-A
tion machinery or via rapid divergence from laterally family is involved in nucleotide excision repair [169],
transferred prokaryotic transcription factors. A similar and appears to have been derived at some point in
scenario appears to be applicable for the four eukaryotic eukaryotic evolution from the functionally similar phage
wHTH families that are not involved in DNA-binding, excisionases. The principal diversification of this assem-
namely the PINT domain, the SNF8 (with two tandem blage appears to have happened early in bacterial evolu-
copies of the wHTH domain), cullin and the DEP domain tion resulting in the two ancient families, MerR and the
families. FTRS b-subunit N-terminal domain. Given that regular
Beyond these multi-member families there are highly 3-standed wHTHs are also found in association with
conserved lineages of 2- or 3- stranded wHTH domains other translation proteins, like the archaeal FTRS-a
that are typically found in single orthologous groups of subunit and SelB, it possible that the prototype of the
proteins, and cannot be linked to any of the larger MerR-like version was derived from such a form
assemblages. Such lineages include wHTHs found in through loss of the initial helix.
the bacterial proteins such as FtsK, DprA and O-6-
methylguanine-DNA alkyltransferase, and the ar- 4.4. Other miscellaneous families of HTH domains
chaeo-eukaryotic proteins like TFIIE (Fig. 3) [30,161].
Distinct from the 2- and the 3-stranded HTH do- In addition to the above-described major assem-
mains are the 4-stranded HTH domains that appear to blages, there are a few ancient HTH families with uncer-
form a separate monophyletic assemblage within the tain affinities. The chief amongst these are the related
wHTH clade (Fig. 5). The main prokaryotic family in ribosomal proteins L11 and S18 (Fig. 2). The former is
this assemblage is the Crp family [36] that has a pan-bac- conserved in all the three super-kingdoms of life and
terial distribution and sporadic presence in few archaea. binds RNA in the L11-stalk structure, which appears
Thus, it seems to represent a bacterial innovation that to go back to the shared ancestral core of the ribosome
was disseminated to the archaea via lateral transfer. [170]. S18 appears to have been derived from L11 in the
Members of this family are typically fused to a C-termi- bacteria. Despite its apparent ancient origins L11 ap-
nal cNMP-binding domain, and appear to have special- pears to be a highly derived version of the HTH. Hence,
ized early on as cyclic nucleotide dependent regulators. notwithstanding certain general similarities with the 4-
The eukaryotes contain a single major family of this stranded wHTHs, it is more likely that L11 has conver-
assemblage, the HSF family (heat shock transcription gently acquired these features early in evolution.
factor), which is present only in the crown group
eukaryotes. In the animals this protein appears to have
spawned two distinct sub-families that are prototyped 5. Proteome-wide demographic trends of HTH domains
by the ETS domain and the IRF domain (interferon reg-
ulator factors) [162,163]. Given their relatively restricted The availability of a large number and phyletic diver-
presence in eukaryotes, it is possible that they have orig- sity of complete genome sequences allows robust estima-
inated through rapid divergence from laterally trans- tion of the general trends in the proteome-wide
ferred prokaryotic versions. A similar scenario could distribution of HTH domains. In order to detect the
be envisaged for the origin of the orphan initiator bind- occurrences of HTH domains in proteomes, position-
ing protein from Trichomonas, which also contains a 4- specific score matrices or sequence profiles were con-
stranded version of the HTH domain. structed for the various distinct families of these domains
The MerR-like assemblage of truncated wHTH do- using seed alignments with diverse representatives. These
mains are derivatives of 3-stranded wHTH domains sequence profiles were then used to iteratively search the
(Fig. 2). The MerR family proper [37] and the related target proteomes with the PSI-BLAST program [42].
wHTH domains present in the DNA-binding region of Alternatively, the alignments were used to generate hid-
the bacterial phenyalanyl tRNA synthetase b-subunit den Markov models, which were similarly used to search
[164] show a pan-bacterial distribution. In bacteria the the proteomes with the HMMER program [42]. Using a
MerR family has vastly proliferated into several distinct combination of these procedures we determined the total
subfamilies, like the SoxR and CueR subfamilies [37]. number of proteins containing HTH domains encoded by
However, most other versions show a more restricted all completely sequenced organisms that were available at
 L. Aravind et al. / FEMS Microbiology Reviews 29 (2005) 231–262 253

the time of the analysis. For prokaryotes, the plot of the provided by a quadratic curve of the form y = a ·
total number of HTH domains against gene number per x2 + b · x; where ÔaÕ and ÔbÕ are constants; r2 = 0.76).
genome is best fitted by the power equation of the form The non-linear scaling of the sigma factors suggests that
y = k · xc (where k and c are constants; r2 = 0.89; see in the more complex genomes the additional genes are
Fig. 7(a)). The r2 of 0.89 for this fit suggests that this ten- distributed amongst several functionally specialized
dency is indeed strongly maintained across a wide diver- gene batteries, which are under the regulation of de-
sity of genomes. This non-linear scaling of HTH voted sigma factors responding to specific situations.
domain numbers with gene number is consistent with re- Interestingly, a few genomes show a significantly greater
cent studies that have suggested that the transcription fac- than expected number of sigma factors (Fig. 7(d)). The
tor counts follow a power equation with respect to gene most striking example is seen in the case of Phytoplasma
number [171]. Given that the HTH domains are the main asteris, which, like other Mycoplasmas, has a highly re-
transcription factors in most of the prokaryotic genomes duced genome with just over 700 genes [172]. Whereas,
it is clear that the trend observed for transcription factors the other Mycoplasmas have only a basal sigma-54, P.
is principally a reflection of the distribution of HTH do- asteris has, in addition to sigma-54, a recent lineage-spe-
mains (Fig. 7). This distribution function suggests that cific expansion of 11 sigma factors that are related to the
as gene number increases, a greater than linear number Bacillus sigma F. Likewise, Bacteroides thetaiotaomicron
of HTH domain regulators are required per gene. and Nitrosomonas show recent lineage-specific expan-
Examination of major architectural classes of HTH sions of ECF-type sigma factors that have given rise
indicates that there is an interesting differential class- to at least 10 closely related paralogous members in their
wise partitioning of the trends. HTH proteins belong- proteomes. In the case of P. asteris there is evidence that
ing to two-component, serine/threonine kinase and the sigma factors may constitute a novel transposon
PTS signaling cascades are almost entirely missing in [173]. While this possibility also exists in the case of
archaea. In the bacteria, where they are abundantly the other bacteria that show a greater than expected
present, their numbers show a linear scaling with re- number of sigma factors, it is likely that some of them
spect to gene count per genome (r2 = 0.8) (Fig. 7(c)) might have been utilized as transcriptional regulators.
[42]. This suggests that each HTH in two-component This potential link between sigma factors and transpo-
and related phosphorylation cascades regulates a fixed son is consistent with the repeated recruitment of
number of target genes, and as the gene numbers grow HTH domains from transposases as specific transcrip-
larger, the regulators increase in direct proportion to tional regulators.
their increase in targets. This is consistent with a model The genomic data from eukaryotes is still not ade-
of evolution in which the two-component systems and quate to discuss general genome-wide trends. However,
their target genes undergo duplication with approxi- the available data suggests that eukaryotes display dif-
mately the same probability as the size of a bacterial ferent tendencies from the prokaryotes. The HTH do-
genome increases in evolution. In contrast to the mains of the homeo and Myb proteins are amongst
two-component systems, the one component systems, the most prominent DNA-binding domains of transcrip-
which chiefly comprise of those HTH domains fused tional regulators in the plant and animal lineages. How-
to specific SMBDs or metabolic enzymes, scaled non- ever their numbers are relatively low in the other
linearly with increase in gene count per genome. The eukaryotic lineages. This suggests that the rise in prom-
best fit for the one component systems was obtained inence of HTH transcription factors may have been a
with the power equation of the form y = k · xc relatively late phenomenon that occurred on multiple
(r2 = 0.76, Fig. 7(b)). This equation suggests that the occasions in the eukaryotic crown group. The parasitic
HTH domains belonging to the one-component system apicomplexa, including those forms that have genome
are likely to be a significant contributor to the over-all sizes comparable to some free-living fungi, have far
power equation-type distribution of the HTH domains. fewer transcription factors in general [174]. The early
This observation implies that as genome size increases branching eukaryote Giardia lamblia has at least 13
a greater than proportional increase in the numbers Myb domains and 2 Bright domains, but apparently
of one-component transcription factors is required for no other representatives of the HTH domains found in
controlling the newly added genes. This tendency might the eukaryotic specific transcription factors (LA unpub-
correlate with the need to regulate specialized groups lished). Furthermore, the scaling of the total number of
of their genes, by combining the distinct inputs sensed transcription factors in eukaryotes with gene counts per
by the effector-binding domains of multiple sets of one- genome is not equivalent with what is observed in the
component transcription factors in the metabolically or prokaryotes. This difference may arise due to two main
organizationally complex bacteria with large genomes. reasons: (1) the emergence of a complex apparatus for
The counts of sigma factors, too on an average, are chromatin-structure regulation might have changed
positively correlated with gene count per genome (Fig. the nature of transcriptional control in eukaryotes. (2)
7(d)), and scale non-linearly with it (the best fit being Most eukaryotic transcription factors are effectively
254 L. Aravind et al. / FEMS Microbiology Reviews 29 (2005) 231–262

Fig. 7. Scaling of HTH domains with gene number per genome. All graphs show a scatter plot of number of proteins with HTH domains in a given
proteome (Y-axis) versus the number of protein-coding genes in that organism (X-axis). (a) The Y-axis is the overall number of proteins with HTH
domains. (b) The Y-axis is the number of predicted one-component system proteins with HTH domains. (c) The Y-axis is number of two-component
system and other phospho-relay system proteins with HTH domains. (d) The Y-axis shows the number of sigma factors. For each graph the best-
fitting trend line along with its r2 value is shown.

down-stream of signaling cascades that communicate the archaeo-eukaryotic clade are ribosomal proteins
from the cell membrane, or the cytoplasm to the nu- (S19AE and S25AE); whereas all the known bacterial
cleus. Hence, there are there are few equivalents of the representatives of this family are specific transcription
genuine prokaryote-type two-component systems in factors (Fig. 5). All other versions of the HTH associ-
the eukaryotes. Additionally, the eukaryotes might also ated with translation and RNA metabolism, such as
extensively employ post-transcriptional control mecha- those found in La/RNA 2 0 phosphatase, SelB and ribo-
nisms, involving regulatory RNAs, resulting in a lower somal protein S10E, appear to have been derived after
dependence on transcriptional regulators [175,176]. A the separation of the archaeo-eukaryotic and bacterial
combination of these factors might account for the dif- clades. The simplest interpretation of these observations
ference in the average number of genes controlled by is that the majority of HTH domains associated with
per transcription factor in eukaryotes and prokaryotes. RNA metabolism settled into their extant functional
niches only after the divergence of the major lineages
from LUCA. Hence, excluding L11, it is likely that other
6. General considerations on the natural history of the HTH domains associated with RNA metabolism in the
HTH fold and implications for the evolution of LUCA performed more generic functions compared to
transcription their extant counterparts. The DNA-binding property
is strongly preserved across diverse lineages and struc-
With the exception of the ribosomal protein L11 no tural variants of the HTH fold, and involves helix-3, de-
bona fide HTH domains with an ancestral RNA-bind- spite the several variations in the details of the
ing role can be confidently traced back to the LUCA. interactions of individual versions of the domain. This
In the HrcA and GntR families the representatives from observation, taken together with the more sporadic
 L. Aravind et al. / FEMS Microbiology Reviews 29 (2005) 231–262 255

distribution of the versions of the domain associated ilies in LUCA performed a generic nucleic-acid-binding
with translation and RNA metabolism, suggests that function. They appear to have been secondarily re-
the ancestor of most of the extant versions of the cruited as specific transcription factors only in the bacte-
HTH, excluding L11, was a DNA-binding protein. Fur- ria, thereby supporting the second scenario. The basal
thermore, the diversification of this domain was poten- transcription factors and ribosomal proteins tend to
tially associated with the emergence of DNA as show a stronger signal of vertical inheritance as com-
genetic material [75,153]. pared to specific transcriptions factors which are prone
While there are HTH domains in the basal transcrip- to rampant gene loss and lateral transfer [32,177]. This
tion factors of both the bacterial (sigma factors) and ar- is not unexpected, given the functional constraints act-
chaeo-eukaryotic (TFIIB, TFIIE, and MBF1) lineages, ing on the basal transcription factors, and might con-
none of these can be considered as being truly ortholo- found the reconstruction of the actual evolutionary
gous [30,32,33]. In contrast, several families of the scenario for the specific transcription factors.
HTH domains in specific transcription factors appear Although the first scenario of basal transcription fac-
to be extensively shared by the bacteria and archaea tors emerging after the specific transcription factors
(Fig. 4). Though several of the prokaryotic families might appear counter-intuitive, detailed analysis of the
shared by bacteria and archaea can be easily explained reconstructed house-keeping functions in the LUCA
as arising from relatively recent lateral transfer between suggest that it is hardly implausible. Earlier studies on
the prokaryotic super-kingdoms, some others like the the DNA replication and chromosome partitioning sys-
MarR, ArsR, YctD, Lrp, HrcA and GntR families ap- tems suggest that the central enzymes of the DNA rep-
pear to show distinct pan-archaeal and pan-bacterial lication apparatus appear to have emerged only after
groups suggesting that they were present in the earliest the split of the archaeo-eukaryotic and bacterial lineages
representatives of each of the super-kingdoms, hence [32,127,178]. Thus, the DNA replication system resem-
potentially go back to the LUCA (Fig. 5). Despite the bles the basal transcription apparatus in its origins.
basal transcription machinery shared with the archaea, More specifically, the absence of an ancestral DNA
the specific transcription factors that are traceable to polymerase and associated replication enzymes in the
the last eukaryotic common ancestor do not belong to LUCA suggest that it probably had a system of replica-
the same families as the specific transcription factors tion involving reverse transcription [127]. However,
seen in the archaea [30]. These patterns raise a profound simpler but completely functional DNA-dependent
conundrum regarding the origin of the phyletic patterns RNApolymerase (DdRP) subunits have been recon-
of HTH domains in the basal and specific transcrip- structed for the LUCA [130,179]. Hence, it is possible
tional regulators of extant organisms. that the LUCA did not use basal transcription factors
Even though several scenarios could in principle ex- and the DdRPs chiefly functioned as enzymes that sup-
plain these patterns, there are only a few parsimonious plied the RNA template for the replication process
alternatives that account for the currently available involving a reverse transcription step. The precursors
data. Given that both the basal transcription machinery of the specific transcription factors might have still func-
and the domain architecture of the RNA polymerase tioned under these circumstances, primarily acting as
catalytic subunits are very different in the bacterial and general repressors that regulated the synthesis of the
the archaeo-eukaryotic lineages [130], one could extrap- RNA template. The basal transcription factors probably
olate that the basal transcription factors arose only after arose only when the genome got organized into multiple
the two great lineages had separated from the LUCA. In tandem operons, each needing its own transcription ini-
this situation, the sharing of the specific transcription tiation signal. This suggestion is consistent with the fact
factors by the archaea and the bacteria could be ex- that the prokaryotic specific transcription factors can
plained in two possible ways: (1) The LUCA had several function equally well with both archaeo-eukaryotic-
specific transcription factors but no basal transcription and bacterial-type basal transcription machinery and
factors. (2) Alternatively, there were neither specific RNA polymerases, despite the numerous differences
nor basal transcription factors in the LUCA and both [180].
types emerged after the lineages separated. However, Irrespective of the scenarios, the HTH domains of
multiple very early lateral transfer events resulted in pro- both the simple tri-helical type (e.g. carbamoylphos-
karyotic lineages sharing a common set of specific tran- phate synthetase and topoisomerase I) and the wHTH
scription factors. This latter scenario is the consistent type (e.g. topoisomerase II family) are present in pro-
with the evidence for extensive lateral transfer between teins that can be confidently traced back to the LUCA.
the two prokaryotic super-kingdoms throughout their Depending on the scenario, there were at least 6–11 dif-
evolution [32,177]. At least in the case of the HrcA ferent HTH domains in the cellular genomes, suggesting
and GntR families, the striking difference in functions that the fold had undergone structural and functional
of the extant bacterial and archaeo-eukaryotic represen- differentiation even before the period of the LUCA.
tatives suggests that the ancestral versions of these fam- Subsequently, in course of prokaryotic evolution, the
256 L. Aravind et al. / FEMS Microbiology Reviews 29 (2005) 231–262

HTH domains rapidly expanded in several prokaryotic tion factors have chiefly emerged through multiple
genomes to rank amongst the folds with highest repre- lineage-specific expansions.
sentation [32,35]. The currently available evidence sug-
gests that the common ancestor of extant eukaryotes
arose later, via an endosymbiotic event involving an 7. General conclusions
archaeal precursor for the nucleus, translation and
secretion apparatus and an a-proteobacterial precursor The HTH domain, one of the best-studied of the
for the mitochondrion. However, neither the mitochon- double-stranded DNA-binding domains, is one of the
drial, nor the nuclear genome, appears to have retained key protein domains in the transcriptional apparatus
the specific transcription factors might have been inher- of all extant organisms. With the ‘‘hindsight’’ of over
ited from the respective prokaryotic precursors. Instead, two decades of investigations since the discovery of the
the principal pan-eukaryotic HTH transcription factor domain we attempt to provide a synthetic overview of
families, like Bright and MYB domains, are only dis- the natural history of the HTH domain from the view-
tantly related to the prokaryotic counterparts. The ori- point of comparative genomics. Despite the HTH being
gin of the eukaryotes saw the emergence of a distinct a rather simple structural scaffold, it is observed to be
nuclear compartment, extensive RNA-dependent post- capable of considerable structural variety and functional
transcriptional gene regulation, a complex chromatin versatility, while still preserving a core set of correlated
structure and the proliferation of enzymatic complexes structure–function features. Most HTH domains, de-
involved in chromatin dynamics, such as the Swi2 ATP- spite their structural diversity, participate in a variety
ases, acetylases and deacetylases [181]. The compart- of functions that depend on their DNA-binding proper-
mentalization of the cell probably rendered the ties. These include their central role in mediating the
prokaryote-type one-component systems ineffective in substrate interactions of various enzymes that operate
the eukaryotes. Furthermore, the regulators of chroma- on DNA, and their role as both basal and specific tran-
tin dynamics, such as the histone deacetylases and asso- scription regulators. Thus, the HTH domains are the
ciated Swi2 ATPases took up the role of transcriptional predominant transcription factors in all prokaryotic
repressors [181], and probably resulted in the ancestral organisms and the more complex eukaryotes, such as
prokaryote-type repressors becoming superfluous. the plants, animals and fungi. Beyond these DNA-
Hence, the origin of the eukaryotes was probably binding functions, the HTH domains have been
accompanied by a massive loss of the prokaryote-type recruited on multiple occasions in a RNA-binding
transcription factors, along with the innovation through capacity and as mediators of protein–protein interac-
rapid sequence divergence of new versions that suited tions. The last universal common ancestor already had
the eukaryotic milieu. Some of the HTH domains inher- anywhere between 6 and 11 distinct versions of the
ited from the ancestral prokaryotic genomes were also HTH domain, which covered much of the structural
reused by the eukaryotes as adaptor modules in signal- diversity, and at least some of the functional diversity
ing systems un-related to DNA-binding or transcription seen in the extant versions of the domain. Though sev-
regulation. However, many versions inherited from the eral families of specific transcription factors are shared
archaea appear to have persisted in the basal transcrip- by the two prokaryotic kingdoms and may even go back
tion machinery, where they were indispensable for tran- to their common ancestor, the HTH proteins in the
scription of the nuclear genome. basal transcription factors do not appear to be ortholo-
Finally, the rise of organizational complexity in gous. This presents an interesting evolutionary conun-
plant, animal and fungal lineages went hand-in-hand drum, whose solution might emerge from new data on
with the emergence of new specific transcriptional regu- alternative transcription and replication systems, like
lators. Some were drawn from pre-existing HTH fami- those in viruses and other selfish elements [75,156].
lies, like the Myb domain, while others like the HSF, The HTH domain occurs frequently in modular pro-
Homeo, Pou, Pipsqueak and Paired families arose teins, whose domain architectures are often correlated
through rapid divergence from different sources. The with the general functional properties of the protein.
HTH domains of transposons provided the source mate- In prokaryotes the dominant domain architecture is
rial for some of these domains, whereas other might the one-component system that combines the HTH with
have diverged rapidly from laterally transferred pro- a sensor domain. While many of the sensor domains of
karyotic transcription factors. Thus, on one hand pro- commonly known one-component systems have been
karyotes appear to share a sizeable common set of characterized previously, several others remain structur-
transcription factors, whose phyletic patterns are chiefly ally and functionally unexplored, and suggest a new
governed by the lateral transfers and gene losses acting direction for exploring the intricacies of biological sen-
over and above a basic signal of vertical inheritance. sors in prokaryotic systems. The extensive use of diver-
On the other hand, the eukaryotes share only a few un- gent HTH domains in protein–protein interactions,
ique ancient DNA-binding domains, and their transcrip- especially in eukaryotes, is another area that might
 L. Aravind et al. / FEMS Microbiology Reviews 29 (2005) 231–262 257

develop further in the future as the actual mechanistic inferred from its homology with cro repressor. Proc. Natl. Acad.
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