SBS 3053 : LECTURE 4

THE MECHANISM OF
TRANSCRIPTION IN BACTERIA
(CHAPTER 6)

6-1
DR MOHD NAZMI ABD MANAP
The flow of genetic information according to the central dogma of molecular
biology. 6-2
 Basic Principles of
Transcription and Translation
• Transcription • During transcription
– Is the synthesis of – The gene determines the
RNA under the sequence of bases along the
direction of DNA length of an mRNA molecule
– Produces messenger DNA Gene 2
RNA (mRNA) molecule
Gene 1
• Translation Gene 3

– Is the actual
synthesis of a DNA strand 3
(template) A C C A A A C C G A G T
5

polypeptide, which
occurs under the TRANSCRIPTION

direction of mRNA mRNA 5

U G G U U U G G C U C A
3
– Occurs on TRANSLATION
Codon

ribosomes 6-3
Protein Trp Phe Gly Ser
Amino acid
• In prokaryotes
– Transcription and translation occur
simultaneously

DNA
TRANSCRIPTION

mRNA
Ribosome

TRANSLATION

Polypeptide

(a) Prokaryotic cell. In a cell lacking a nucleus, mRNA

produced by transcription is immediately translated
without additional processing.
6-4
• In eukaryotes
– RNA transcripts are modified before
becoming true mRNA

Nuclear
envelope

TRANSCRIPTION DNA

Pre-mRNA
RNA PROCESSING

mRNA

Ribosome

TRANSLATION
(b) Eukaryotic cell. The nucleus provides a separate
Polypeptide compartment for transcription. The original RNA
transcript, called pre-mRNA, is processed in various
ways before leaving the nucleus as mRNA.

6-5
 OBJECTIVE

• Topics :
 Classification of RNA Molecules
 Transcription Phases
 Transcription vs Replication
 RNA Polymerase Structure
 Promoters
 Transcription Initiation
 Elongation
 Termination of Transcription

6-6
 CLASSIFICATION OF RNA MOLECULES
mRNA – messenger RNA : rRNA – ribosomal RNA :
-bring information from the DNA -80% of total RNA
-Translated into protein -Component of ribosome
-Not stable -Involve in protein synthesis
-stable

RNA molecules

SnRNA – small nuclear RNA : tRNA – transfer RNA :

- Involve in processing RNA -Has specific 2ºand 3º structure
-Binds amino acid to mRNA
-Long lifespan
-Involve in protein synthesis
6-7
 TRANSCRIPTION
• Is the generation of an mRNA copy of
the genetic information coded in the
DNA

• Transcription follows the same base-

pairing rules as DNA replication
– Remember U replaces T in RNA

• The enzyme directing transcription is

called RNA polymerase
6-8
 TRANSCRIPTION PHASES

Transcription
occurs in
three
phases:

1. Initiation
2. Elongation
3. Termination

6-9
 Synthesis of an RNA Transcript
Promoter
Transcription unit

• The stages of
5 3
3 5
DNA
Start point
RNA polymerase 1 Initiation. After RNA polymerase binds to
transcription the promoter, the DNA strands unwind, and
the polymerase initiates RNA synthesis at the
start point on the template strand.
are 5
3
3
5

Unwound RNA Template strand of DNA

DNA transcript
2 Elongation. The polymerase moves downstream, unwinding the
DNA and elongating the RNA transcript 5  3 . In the wake of
– Initiation Rewound transcription, the DNA strands re-form a double helix.

RNA

– Elongation 5
3
5
3
3
5

RNA

– Termination transcript
3 Termination. Eventually, the RNA
transcript is released, and the
polymerase detaches from the DNA.

5 3
3 5

5 3

Completed RNA transcript

6-10
 INITIATION
• RNA polymerase recognizes a region,
the promoter, which lies just upstream of
gene
• Polymerase binds tightly to promoter
causing localized separation of the two
DNA strands
• Polymerase starts building the RNA
chain adding ribonucleotides
• After several ribonucleotides are joined
together the enzyme leaves the promoter
and elongation begins
6-11
 ELONGATION
• RNA polymerase directs binding of
ribonucleotides in the 5’ to 3’ direction

• Movement of the polymerase along the

DNA template causes the “bubble” of
separated DNA strands to move also

• As DNA transcription passes, the two

DNA strands reform the double helix
6-12
 TERMINATION
• Analogous to the initiating activity of
promoters, there are regions at the other
end of genes that serve to terminate
transcription
• These terminators work with the RNA
polymerase to loosen the association
between RNA product and DNA template
• As a result, the RNA dissociates from
the RNA polymerase and the DNA and
transcription stops
6-13
 TRANSCRIPTION LANDMARKS
• RNA sequences are transcribed from 5’ to
3’, left to right
• Translation occurs 5’ to 3’ with ribosomes
reading the message 5’ to 3’
• Genes are written so that transcription
proceeds from left to right
• The gene’s promoter area lies just before
the start area, said to be upstream of
transcription
• Genes are therefore said to lie
downstream of their promoters 6-14
 SUMMARY
• Transcription takes place in three stages:
– Initiation
– Elongation
– Termination
• Initiation involves binding RNA polymerase
to the promoter, local melting and forming
the first few phosphodiester bonds
• During elongation, the RNA polymerase
links together ribonucleotides in the 5’ to 3’
direction to make the rest of the RNA
• In termination, the polymerase and RNA
product dissociate from the DNA template
6-15
 TRANSCRIPTION VS DNA
REPLICATION
• Differences • Similarities
1. RNA polymerase only 1. 5’-3’ direction
makes one RNA 2. Many protein involved
strand during 3. Phase – initiation,
transcription, it elongation and
copies only one DNA termination
strand in a given
gene; replication 4. Starts and stops at
copies both strands specific sites
2. DNA melting is
limited and transient
during transcription,
but the separation is
permanent in 6-16
replication
 RNA POLYMERASE STRUCTURE
SDS-PAGE of RNA polymerase from E. coli
had shown several subunits - 2a, 1b, 1 b’, 1s &
1w.
– 2 very large subunits are b (150 kD) and b’
(160 kD)
– Sigma (s) at 70 kD
– Alpha (a) at 40 kD – 2 copies present in
holoenzyme
– Omega (w) at 10 kD
• Appears to play a role in enzyme assembly
6-17
Structure of RNA polymerase

6-18
Structure of RNA polymerase

6-19
 RNA POLYMERASE STRUCTURE
& FUNCTION
holoenzyme - 2a, 1b, 1 b’, & 1s.

– Subunits a & b (tetramer) form the core

enzyme

– Subunits b provide the catalytic basis and

active site for transcription

– Subunit s70 needed for initiation transcription

6-20
 PROMOTERS
• Nicks and gaps are good sites for RNA
polymerase to bind non-specifically

• Presence of the s-subunit permitted recognition

of authentic RNA polymerase binding sites

• Polymerase binding sites are called promoters

• Transcription that begins at promoters is

specific, directed by the s-subunit
6-21
 RNA POLYMERASE BINDING
RNA polymerase holoenzyme binds DNA loosely
at first
– Binds at promoter initially
– Scans along the DNA until it finds one

• Complex with holoenzyme loosely bound at the

promoter is known as closed promoter complex
as DNA is in a closed ds form

• Holoenzyme can then melt a short DNA region

at the promoter to form an open promoter
complex with polymerase bound tightly to DNA
6-22
 POLYMERASE/PROMOTER BINDING
• Holoenzyme binds DNA
loosely at first

• Complex loosely bound

at promoter = closed
promoter complex,
dsDNA in closed form

• Holoenzyme melts DNA

at promoter forming open
promoter complex -
polymerase tightly bound 6-23
 CORE PROMOTER ELEMENTS
• There is a region common to bacterial promoters
described as 6-7 bp centered about 10 bp upstream of
the start of transcription = -10 box (TAtAaT)
• Another short sequence centered 35 bp upstream is
known as the -35 box (TTGACa)
• Comparison of thousands of promoters has produced a
consensus sequence for each of these boxes

6-24
 UP ELEMENT
• Some very strong promoters have an
additional element (UP element) upstream
the core promoter – makes these
promoters even more attractive to RNA
polymerase - The rrnB P1 gene Promoter

6-25
 TRANSCRIPTION INITIATION
• Transcription initiation was assumed to
end as RNA polymerase formed 1st
phosphodiester bond

• Very small oligonucleotides (2-8 nt long)

are made without RNA polymerase leaving
the DNA promoter - Abortive transcripts

• 10 nt or more – transcripts stable to

hybridize with the template & elongate
6-26
 STAGES OF TRANSCRIPTION
INITIATION
• Formation of a closed
promoter complex
• Conversion of the closed
promoter complex to an
open promoter complex
• Polymerizing the early
nucleotides – polymerase
at the promoter
• Promoter clearance –
transcript becomes long
enough to form a stable
hybrid with template
6-27
 THE FUNCTIONS OF s
• The s subunit RNA polymerase is an “initiation
factor” - causes tight binding between RNA
polymerase and promoters
• There are several different sigma factors in E.
coli that are specific for different sets of genes
• Sigma factor functions to ensure that RNA
polymerase binds stably to DNA only at
promoters (binds tightly – causes open
promoter complex)
• Sigma destabilized nonspecific binding to non-
promoter DNA
• Sigma stabilizes specific binding to promoter
DNA 6-28
 SIGMA MAY NOT DISSOCIATE
FROM CORE DURING
ELONGATION
• The sigma factor changes its relationship
to the core polymerase during elongation

• It may not dissociate from the core

• May actually shift position and become

more loosely bound to core
6-29
 LOCAL DNA MELTING AT THE
PROMOTER
• Binding to promoter – RNA polymerase causes
melting near the transcription start site
(transcription bubble)

• Transcription bubble moves with the

polymerase exposing the template strand – can
be transcribed

• size of the DNA transcription bubble in

complexes where transcription was active was
found to be 17-18 bp
6-30
 STRUCTURE AND FUNCTION OF s
• Genes encoding a variety of s-factors
have been cloned and sequenced

• There are striking similarities in amino

acid sequence clustered in 4 regions

• Conservation of sequence in these

regions suggests important function

• All of the 4 sequences are involved in

binding to core and DNA 6-31
Homologous Regions in
Bacterial s Factors

6-32
 E. coli s70
• Four regions
of high
sequence
similarity are
indicated

• Specific areas
that recognize
the core
promoter
elements, -10
box and –35
box are notes 6-33
 Region 1

• Role of region 1 appears to be in

preventing s from binding by itself to
the DNA

• This is important as a free s binding to

promoters could inhibit holoenzyme
binding and thereby inhibit transcription

6-34
 Region 2
• This region is the most highly conserved
of the four

• There are four subregions – 2.1 to 2.4

• 2.4 recognizes the promoter’s -10 box

• appears to be a-helix
6-35
 Regions 3 and 4
• Region 3 is involved in both core and
DNA binding

• Region 4 is divided into 2 subregions

– This region seems to have a key role in
promoter recognition
– Subregion 4.2 contains a helix-turn-helix
DNA-binding domain and appears to govern
binding to the -35 box of the promoter
6-36
 SUMMARY

• Comparison of different s gene sequences

reveals 4 regions of similarity among a wide
variety of sources

• Subregions 2.4 and 4.2 are involved in

promoter -10 box and -35 box recognition

• The s-factor by itself cannot bind to DNA, but

interaction with core unmasks a DNA-binding
region of s
6-37
 Role of a-Subunit in UP
Element Recognition
• RNA polymerase itself can recognize an
upstream promoter element, UP element

• While s-factor recognizes the core

promoter elements, what recognizes the
UP element?

• It appears to be the a-subunit of the core

polymerase 6-38
 ELONGATION
• After transcription Elongation Non-template

initiation is strand of DNA

RNA nucleotides

accomplished, core RNA

polymerase
polymerase
continues to elongate A
T C C A A
3
the RNA 3 end
U
A E G C A
5

• Nucleotides are T A G G T T

added sequentially, 5
Direction of transcription
Template
one after another in (“downstream”)
strand of DNA

the process of Newly made

elongation RNA

6-39
 FUNCTION OF THE CORE
POLYMERASE
• Core polymerase contains the RNA
synthesizing machinery

• Phosphodiester bond formation involves

the b- and b’-subunits

• These subunits also participate in DNA

binding
6-40
 TOPOLOGY OF ELONGATION
• Elongation of transcription involves
polymerization of nucleotides as the RNA
polymerase travels along the template DNA

• Polymerase maintains a short melted region of

template DNA

• DNA must unwind ahead of the advancing

polymerase and close up behind it

• Strain introduced into the template DNA is

relaxed by topoisomerases 6-41
 TERMINATION OF TRANSCRIPTION
• When the polymerase reaches a
terminator at the end of a gene it falls off
the template and releases the RNA

• There are 2 main types of terminators

– Intrinsic terminators function with the RNA
polymerase by itself without help from other
proteins

– Other type depends on auxiliary factor called

r, these are r-dependent terminators 6-42
 RHO-INDEPENDENT TERMINATION

• Intrinsic or r-independent termination

depends on terminators of 2 elements:
– Inverted repeat followed immediately by
– T-rich region in nontemplate strand of the
gene

• An inverted repeat predisposes a

transcript to form a hairpin structure

6-43
 INVERTED REPEATS AND HAIRPINS

• The repeat at right is

symmetrical around
its center shown with
a dot

• A transcript of this
sequence is self-
complementary
– Bases can pair up to
form a hairpin as seen
in the lower panel 6-44
 Rho-Independent termination
• The polymerase paused
with the formation of
hairpin

• formed hairpin will

destabilized the
hybridization of
polymerase-DNA, thus lead
to termination
• The hairpin formed in
synthesized RNA not in
DNA
6-45
 RHO-DEPENDENT TERMINATION

• Rho effects chain elongation

• Rho causes production of shorter

transcripts

• Rho releases RNA transcripts from the

DNA template

6-46
 MECHANISM OF RHO
• No string of T’s in the
r-dependent
terminator, just
inverted repeat to
hairpin
• Binding to the growing
transcript, r follows
the RNA polymerase
• It catches the
polymerase as it
pauses at the hairpin
• Releases transcript
from the DNA-
polymerase complex
by unwinding the RNA-
DNA hybrid 6-47
SUMMARY : TRANSCRIPTION
• Is the generation of an mRNA copy of the
genetic information coded in the DNA

• Transcription follows the same base-pairing

rules as DNA replication
– Remember U replaces T in RNA

• The enzyme directing transcription is called

RNA polymerase
• 3 steps :
 Initiation
 Elongation
 Termination
6-48
 TRANSCRIPTION : INITIATION
Holoenzyme bind to a promoter (-10 & -35) in DNA

With the help of s-factor

Form closed promoter complex

A few base of DNA melting – form open promoter complex

Unwinding of the 2 strands of DNA – providing template strands free to

base-pair with incoming ribonucleotide

The formation of phosphodiester bonds between the first few

ribonucleotides in the RNA chain

Synthesis of the first 8-9 ribonucleotides (The holoenzyme remains bound at

the promoter region) & 8-9 ribonucleotides released – called abortive transcript

Abortive synthesis stops once chains of 10 or more ribonucleotides have been

synthesized – transcript becomes long enough to form a stable hybrid with
6-49
template
 TRANSCRIPTION : ELONGATION
RNA pol has begun to move downstream from the promoter – elongation start

With the help of core enzyme – 2 α, β, β’

The extension of RNA chains takes place within the transcription bubble
(a locally unwound segment of DNA = 17-18 bp)

RNA pol continuosly unwinds the dsDNA ahead of the polymerization site &
rewinds the cDNA strand behind the polymerization site as it moves along the
double helix

The RNA chain is displaced from the DNA template strand as RNA pol moves
along the DNA mol – the region of transient base-pairing between the
growing chain & the DNA template strand is very short, only 3 bp
(hybridization of RNA & DNA template) 6-50
 TRANSCRIPTION : TERMINATION

rho-independent terminator =
inverted repeat & T rich  rho-dependent terminator –
form hairpin structure inverted repeat

rho binds to the growing RNA chain and

moves 5’ to 3’ along the RNA  like
chasing the RNA pol mol

When RNA pol slow down @ pause (meet

an inverted repeat)

rho catches up with the RNA pol & pulls the

RNA chain from the transcription bubble 
transcription terminates 6-51