Lehninger

Principles of Biochemistry

26. RNA metabolism

 DNA RNA

Sugar 2’-deoxyribose ribose

Base Thymine Uracil

Strand Double stranded Single stranded

Potential for much greater structural diversity

than DNA
Two functions of RNA
1. The storage and transmission of information
DNA  RNA  protein
transcription translation
2. Catalysis : ribozyme (catalytic RNA)
3. Components of ribosomes
4. Regulation of gene expression
All RNA molecules except the RNA genomes of certain viruses are derived
from information stored in DNA

mRNA
DNA RNA tRNA
Transcription
rRNA

5’ 3’
3’ 5’
Transcription

5’ mRNA

aa aa
Translation
tRNA

Polypeptide
Ribosome: rRNA + proteins
Prokaryotic 70S ribosome :
30S subunit : 16S rRNA + 21 proteins
50S subunit : 5S rRNA + 23S rRNA + 31 proteins
Eukaryotic 80S ribosome
40S subunit : 18S rRNA + 33 proteins
60S subunit : 5S, 5.8S, 28S rRNAs + 49 proteins
DNA-dependent synthesis of RNA
Transcription 과 replication 의 공통점
1. Direction of synthesis (5’3’)
2. Initiation, elongation, termination phase
3. A template is required

Transcription 과 replication 의 차이점

1. Transcription does not require a primer
2. Only one DNA strand serves as a template in transcription
RNA is synthesized by RNA polymerases
DNA-dependent RNA polymerase
1. DNA template is required
2. Four ribonucleotides (ATP, GTP, UTP, CTP), Mg2+
3. RNA synthesis in the 5’3’ direction
(NMP)n + NTP (NMP) n+1 + PPi
RNA lengthened RNA
Transcription by RNA Polymerase in
E. coli
4. Only one of the two DNA strands serves as a template
Template strand, nontemplate strand (coding strand)
5. Each nucleotide in the newly formed RNA is selected by Watson-Crick base-
pairing interactions
A-U, G-C
6. RNA polymerase does not require a primer to initiate synthesis
7. The first residue of RNA has the 5’-triphosphate group
8. Hybrid RNA-DNA double helix : 8 bp
9. Transcription bubble : ~17 bp
10. Elongation of a transcript by E. coli RNA polymerase proceeds at a rate of 50 –
90 nt/sec
 Overwound DNA

(~17 nt)
DNA-dependent RNA polymerase of E. coli
Quaternary structure : ’ (holoenzyme)
 ’ (core enzyme)
 subunit : promoter recognition
Molecular weight 에 따라  , etc.
RNA polymerase lacks 3`5` proof-reading function
The active site is formed by the  and ’ subunits
Promoter
The site where the RNA polymerase binds to and directs the initiation of
transcription
RNA synthesis is initiated at promoters
70 promoter : -10 region + -35 region + UP (upstream promoter) element
-10 and –35 regions : interaction sites for the 70 subunit
UP element : -40 - -60, AT-rich region
The UP element is bound by the a-subunit of RNA pol,
found in the promoters of highly expressed genes

어떤 gene 의 promoter 가 – 35, -10 region 의 consensus sequence 와 비슷할수록

 strong promoter
Weak promoter  activator protein is required
Initiation
1. Closed complex formation
2. Open complex formation
3. Initiation of transcription
4. Promoter clearance

R+P RPc RPo

isomerization -10 - +3

8-9 nt RNA
The first nucleotide is generally
ATP or GTP NusA

Elongation

5’
Transcription Initiation and Elongation in E. coli
Gene expression is regulated largely at the transcriptional level

Gene on DNA mRNA protein

Transcriptional regulator
1. Activator : a regulatory protein that activates transcription by
facilitating RNA pol binding or open promoter formation
2. Repressor : a regulatory protein that inhibits transcription by
blocking RNA pol binding or open promoter formation
Termination of transcription in E. coli
1. Rho-independent termination
2. Rho-dependent termination

1. Rho-independent termination
Rho protein is not needed
Palindromic sequence + a short string (6-8 nt) of uridylates at 3’end

Terminator
The rU-dA base pair is weaker than rU-rA and dT-dA

2. Rho-dependent termination
Rho protein: homohexamer, RNA-DNA helicase activity
Rho-binding site on RNA (60-100 nt, rich in CA)
Rho-dependent terminator : no UUUUUU, palindromic sequence
Rho-independent terminator

6-8 nt
Termination of
Transcription in E. coli
by -Independent
Pathway
Termination of Transcription in
E. coli by -Dependent Pathway
 Eukaryotic cells have three kinds of nuclear RNA polymerases
1. RNA pol. I : synthesis of preribosomal RNA (18S, 5.8S, 28S rRNAs)
In nucleolus

2. RNA pol. II : synthesis of mRNA

3. RNA pol. III : synthesis of tRNAs, 5S rRNA, and some small RNAs)

In nucleoplasm
Type II promoter
TATA box (TATAAA) at –30 region
Inr (initiator) sequence near the transcription start site
RNA polymerase II requires many other protein factors for its activity
RNA pol. II : Enzyme consisting of 12 subunits
Transcription factors are required for the formation of the
active transcription complex
Basal (general) transcription factors : Required at every type II promoter
e.g., TFIIA, B, D, E, F, H

TFIID : TBP (TATA box binding protein) + at least 8 TAFs

TBP associated factor

 RBP1 ’

RBP2 
RBP3, 11 2

Carboxyl terminal domain of RBP1

Many repeats of (YSPTSPS):
phosphorylation site
27 repeats in yeast, 52 in mouse and
human
Transcription by RNA polymerase II
1. Initiation
-Closed complex formation : assembly of RNA pol. and transcription factors
at a promoter
-Open complex formation
-Promoter clearance
2. Elongation
3. Termination
Assembly of RNA polymerase and transcription
factors at a promoter
Binding of TFIID to the TATA box
TFIIB binding (with help of TFIIA, not essential)
Binding of TFIIF-RNA pol. II complex
Binding of TFIIE and TFIIH
Formation of the closed complex (preinitiation
complex)
TFIIF : Targeting RNA pol. II to its promoter by interacting with TFIIB and by
reducing the nonspecific binding
TFIIH : DNA helicase activity  formation of open complex
Kinase activity  phosphorylation of RNA pol. II at several places
in the carboxyl-terminal domain of polymerase’s largest subunit
RNA strand initiation and promoter clearance
Phosphorylation of RNA pol. II by TFIIH conformational change in the overall
complex  initiation of transcription

During synthesis of the initial 60 to 70 nucleotides of RNA, first TFIIE and then
TFIIH are released

Elongation and termination

During elongation, elongation factors greatly enhance the polymerase activity
The termination mechanism is unknown
After termination of transcription, RNA pol. II is dephosphorylated and recycled
Regulation of RNA pol. II activity
Interaction of a wide variety of regulatory proteins with the preinitiation
complex

Regulatory proteins-transcription factors (e.g., TFIID)

Regulatory proteins-RNA polymerase II
Diverse functions of TFIIH
1. Unwind DNA at promoter  helicase activity
2. Phosphorylate RNA polymerase  kinase activity
3. Helicase in the nucleotide excision repair system (Fig. 25-23)
Inhibitors of DNA-dependent RNA polymerase
1. Actinomycin D, acridine  intercalator
Inhibition of the elongation of RNA strands by RNA polymerase
Inhibitors for both prokaryotic and eukaryotic RNA polemerases
-GG-
-CC-
2. Rifampicin
Inhibitor of bacterial RNA polymerase
Binding to the b-subunit of RNA polymerase
Prevent the promoter clearance step

3. -amanitin
Inhibitor of RNA polymerase II and at high conc. RNA polymerase III
 RNA polymerase I and III recognize distinct promoters, using distinct sets
of transcription factors, but still require TBP

Type I promoter

TBP+3 TAFs
RNA processing
In eukaryotic genes (in nucleus)
Intron: noncoding region that interrupts the coding
region of a gene
Exon: the coding region

Genes of higher eukaryotes : intron > exon (in length)

Intron
1. The introns transcribed into RNA are removed by splicing
2. In bacteria generally no introns are present
3. In 1977 Phillip Sharp and Richard Roberts discovered
introns
4. The vast majority of genes in vertebrates contain introns
5. Many genes in the yeast Saccharomyces cerevisiae lack
introns
6. Introns are also found in a few bacterial and
archaebacterial genes
RNA splicing
Four classes of introns
1. Group I intron : primary transcripts of some nuclear,
mitochondrial, and chloroplast genes encoding rRNAs,
mRNAs, and tRNAs
2. Group II : primary transcripts of mitochondrial and
chlroplast mRNAs in fungi, algae, and plants
3. Spliceosomal intron : nuclear mRNA primary
transcripts
4. Group IV : certain tRNA
1. Group I splicing
Requires a guanosine, GMP, GDP, or GTP
The 3’-hydroxyl group of guanosine or guanine
nucleotides serves as a nucleophile  formation of
3’,5’-phosphodiester bond with the 5’ end of the
intron
The 3’-hydroxyl group of the upstream exon acts as a
nucleophile and attacks the 5’-phosphate group of
the next exon
transesterification

transesterification
2. Group II splicing
Similar to Group I splicing
The first nucleophile : the 2’-hydroxyl group of an adenylate
residue within the intron  formation of a branched lariat
structure

Group I and II introns are “self-splicing” :

No protein enzymes are involved  RNA has catalytic
function  ribozyme
No ATP is required
3. Splicing of spliceosomal introns
Similar to Group II splicing : formation of the lariat structure
Spliceosome made up of small nuclear ribonucleoproteins
(snRNP, snurp) are required
snRNP : snRNA (100-200 nt) + proteins

U1
U2
U4
U5
U6

snRNP : U1…U6 snRNP

The RNAs and proteins in snRNPs are highly conserved
in eukaryotes from yeast to human

Spliceosome : five snRNAs + ~50 proteins

ATP is required for assembly of the spliceosome
Internal rearrangement of spliceosome
4. Group IV splicing
ATP and endonuclease are
required

phosphatase
Eukaryotic mRNAs undergo additional processing
1. Capping
Most mature eukaryotic mRNAs have a 5’-cap
5’-cap : a residue of 7-methylguanosine linked to the 5’-
terminal residue of the mRNA through a 5’,5’-
triphosphate linkage
Function
Binding of the mRNA to the ribosome to initiate
translation through a cap-binding protein
Protection of mRNA from degradation

Transcription 과정중에 약 30 nt 의 mRNA 가 합성되기 전에

일어난다
SAM
2. Polyadenylation
Most eukaryotic mRNAs have a string of 80 to 250
adenylate residues called the poly(A) tail

Function
Protection of mRNA from degradation

Polyadenylation enzyme complex:

Endonuclease + polyadenylate polymerase + other
proteins
Cleavage site
10 to 30 nt downstream of 5’AAUAAA3’
20 to 40 nt upstream of G,U-rich sequence

Polyadenylate polymerase
RNA + nATP  RNA-(AMP)n + nPPi
n=80-250
Multiple products are derived from one gene by
differential RNA processing
In many cases
1 primary transcript  1 mature mRNA  1 polypeptide
RNA processing translation
1 primary transcript
Poly(A) site choice, alternative splicing
More than 1 mature mRNAs

More than 1 polypeptides

RNA editing
1. Deamination (cytidine deaminase, adenosine deaminase acting on RNA)

Human apolipoprotein gene

Cytidine deaminase
NA editing
Insertion and deletion

Trypanosome
Mitochondrial gene
ADAR (adenosine deaminase acting on RNA)
Poly(A) site choice Alternative splicing
In rats

Calcitonin gene related peptide

Posttranscriptional processing of rRNA

1. In prokaryotes
Primary transcript : 30S preribosomal RNA (in E. coli 7 pre rRNA)

Upseudo U, dihydro U
i) Methylation at specific bases or 2’-OH
ii) Cleavage by RNase III, P, E
iii) Trimming by specific nucleases

16S rRNA + 23S rRNA + 5S rRNA + tRNAs

 (1 or 2)

tRNA

1: RNase III, 2: RNase P, 3: Rnase E

2. In eukaryotes

DNA
Transcription by RNA polymerase I

Primary transcript : 45S preribosomal RNA

i) Methylation on the 2’-OH groups of ribose, Upseudo U
ii) Cleavage by nucleases

18S, 5.8S, 28S rRNAs

Small nucleolar RNAs (snoRNA) are required

Component of snoRNPs
60-300 nt
Many are encoded within the introns of other genes
DNA 5S rRNAs
Transcription by RNA polymerase III
Posttranscriptional processing of tRNAs
Most cells have 40-50 distinct tRNAs
1. In prokaryotes and eukaryotes
tRNAs are contained in the 30S pre-rRNA transcript in prokaryotes
Or
Two or more different tRNAs are contained in a single primary transcript

Primary transcript RNase III

tRNA1 tRNA2 tRNA3

1. Formation of the mature 5’-end
By RNaseP (endonuclease)
125 kDa M1 RNA (catalytic subunit, 377 nt)
17.5 kDa protein (stabilization of the RNA, facilitation of the RNA
function)
2. Formation of the mature 3’-end
3’ cleavage by one or more nucleases including exonuclease called
RNaseD
Addition of CCA-3’ by tRNA nucleotidyltransferase

3. Base modification
Methylation
Deamination
Reduction
Pseudouridine 의 경우 uracil 내의 N-glycosylic bond 형성위치 변화
4. Splicing in some eukaryotic tRNAs (14 nt intron)
 D loop T loop

Anticodon loop

D loop : several dihydrouridine

Anticodon loop : bind to mRNA in translation
T loop : 5’TC3’
 Adenosine

5-methylcytosine

cytosine
Special-function RNAs undergo several types of processing
1. snoRNA

Many snoRNAs are derived from introns  binding of snoRNP proteins

 Trimming by nucleases

2. snRNA

Synthesized as the pre-snRNA (RNA pol II)  trimming by nucleases

 Base modification (2’-OH methylation, uridine  pseudoU)

3. Micro RNA: regulatory RNA, about 22 nt long

How miRNAs are
Processed to Prevent
Translation
 Ribozymes
1. Rnase P
2. Group I intron
3. Hammerhead ribozyme: endoribonuclease found in virusoids
4. U2, U5, U6 snRNAs in RNA splicing
5. 23S rRNA in prokayotic ribosomes

Virusoids are circular single-stranded RNAs dependent on plant viruses for

replication and encapsidation The genome of virusoids consist of several hundred
nucleotides and does not code for any proteins.

Viroid: no capsid
 Comparison of RNAs in prokaryotes

mRNA rRNA tRNA

5’-end 5’-PPP 5’-P 5’-P

Stability Not stable stable stable

Translated to yes no no
proteins
Cellular mRNAs are degraded at different rates

Gene expression
Gene on DNA  mRNA  protein
transcription translation

The concentration of mRNA depends on

1. The rate of synthesis
2. The rate of degradation

The stability of mRNA

Eukaryotic mRNA >> prokaryotic mRNA
RNA is degraded by ribonucleases

A major degradative pathway in eukaryotic cells

1. Shortening of the poly(A) tail
2. Decapping of the 5’ end
3. Degradation of the RNA in the 5’3’, 3’5’ direction
Major in higher eukaryotes(exosome)

5’ 3’ In prokaryote
1. Endoribonuclease
2. 3’5’
exoribonuclease

The hairpin structure and the poly(A) tail of RNA confer stability against
degradation
Polynucleotide phosphorylase

(NMP)n + NDP (NMP)n+1 + Pi

RNA lengthened RNA

Substrate : Ribonucleoside 5’-diphosphate

No template is required
Reverse reaction : degradation of RNA in the 3’5’ direction

The probable function of this enzyme in the cell is the degradation of RNA to
ribonucleoside diphosphate
RNA-dependent synthesis of RNA and DNA

Reverse
transcriptase

RNA replicase
Reverse transcriptase : RNA-dependent DNA polymerase

Retrovirus : the RNA viruses that contain reverse transcriptase

 Viral RNA

Viral DNA

Provirus
Retroviral Infection of a Mammalian Cell and
Integration into Host Chromosome
Retroviral genome typically have three genes
Long terminal repeat sequence (LTR)
At both ends of the linear RNA genome
A few hundred nt long
Integration of the viral DNA into the host DNA
Contains the promoters for viral gene expression
Reverse transcriptase
Zn2+ dependent
Single-stranded RNA  double stranded DNA
RNA-dependent DNA synthesis (5’3’)
RNA degradation : RNase H : degradation of the RNA part in RNA-DNA
DNA-dependent DNA synthesis (5’3’)
Template is required
Primer is required
in viral infection, lysyl-tRNA serves as a primer
No 3’5’ proofreading exonuclease activity

Intron 이 없는 eukaryotic gene 을 얻을려면

Mature mRNA cDNA (complementary)
Reverse transcription
Reverse transcriptase

cDNA
Retroviruses causes cancer and AIDS
Among retroviruses,
RNA tumor viruses contain an oncogene that can cause cancer
e.g., Rous (avian) sarcoma virus

oncogene
HIV (human immunodeficiency virus)
Cause AIDS (acquired immune deficiency syndrome)
HIV kills many of the cells it infects (especially T lymphocytes)
The reverse transcriptase of HIV is very error-prone
Retrotransposon:
Eukaryote 에서 발견되는 retroviral provirus 와 구조가 유사한 transposon
Reverse transcriptase gene
LTR sequences at both ends
Most eukaryotic transposons belong to this class
No env gene  no formation of viral particles (defective provirus)
Telomerase : RNA (~150 nt) + protein

3’

T-loop

Telomere repeat binding factor

Telomerase is a specialized reverse transcriptase

Telomere
5’(TxGy)n

X, Y = 1-4

Telomeres have the 3’-

overhang

5’
5’ 3’
RNA replicase (RNA-dependent RNA polymerase)
4 subunits (RNA replicase of most bacteriophages)
65 kDa protein : catalytic subunit, viral replicase gene product
Tu, Ts : host translation elongation factors
S1 : 30S ribosomal subunit 의 protein
New RNA strand synthesis proceeds in the 5’3’ direction
RNA template is required (template specificity)
No primer is required
No proofreading function
In RNA bacteriophage (f2, MS2, R17, and Q), influenza virus : single-stranded
RNA genome