Chapter 6

The Mechanism of
Transcription in
Bacteria
Course Objectives:
 Basic principles of transcription
and its control in bacteria.
 RNA polymerase and its interaction
with sigma factor as well as
activator and repressor proteins.
 Different stages of transcription
including initiation, elongation and
termination.
 DNA elements involved in
transcriptional regulation.
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Student Expected Learning Outcomes
1. Discuss Structure and function of bacterial RNA
polymerase.
2. Identify the different bacterial core promoter
elements and discuss the important role played by
sigma factor in their recognition.
3. Explain how RNA polymerase is recruited to the
promoter region, and identify the different stages of
transcriptional regulation.
4. Understand how sigma factor contributes to
transcriptional initiation, and how transcriptional
elongation proceeds followed by either rho-dependent
or rho-independent termination.

6-2
 6.1 RNA Polymerase Structure
 By 1969 SDS-PAGE of RNA
polymerase from E. coli had
shown several subunits.
 2 very large subunits are β (150
kD) and β’ (160 kD)
 Sigma (σ) at 70 kD
 Alpha (α) at 40 kD – 2 copies
present in holoenzyme
 Omega (ω) at 10 kD
• Was not clearly visible in SDS-
PAGE, but seen in other  RNA polymerase
experiments holoenzyme (Fr1): β’,β, ,
• Not required for cell viability 2,and .
or in vivo enzyme activity
 Core RNA polymerase
• Appears to play a role in (Fr4): β’,β, and α2.
enzyme assembly 6-3
 Sigma as a Specificity Factor
 Core enzyme without the σ subunit could not
transcribe viral DNA, yet had no problems
with highly nicked calf thymus DNA.
 With  subunit, the holoenzyme worked
equally well on both types of DNA.

6-4
 Summary
 The key player in the transcription process is
RNA polymerase.
 The E. coli enzyme is composed of a core,
which contains the basic transcription
machinery, and a -factor, which directs the
core to transcribe specific genes (T4 genes
called immediate early genes).
 Bautz and colleagues showed by hybridization
to properly transcribed T4 phage RNA that
the core polymerase lacks specificity
compared to holoenzyme.

6-5
 6.2 Promoters
 Why was the core RNA polymerase
capable of transcribing nicked DNA in
the previous table?
 Nicks and gaps are good sites for RNA
polymerase to bind nonspecifically.
 The presence of the σ-subunit permits
recognition of authentic RNA polymerase
binding sites called promoters.
 Transcription that begins at promoters
is specific, directed by the σ-subunit.
6-6
 Binding of RNA Polymerase to
Promoters
 How tightly does core enzyme v.
holoenzyme bind DNA?
 Hinckle and Chamberlin incubated 3H-
labeled T7 phage DNA with either
holoenzyme or core polymerase - then
they added excess cold DNA – Passed
reactions thru nitrocellulose filter.
 Experiment measures dissociation rate
of the polymerase-DNA complex.
 Holoenzyme binds filters tightly (t1/2= 30-
60hrs)
 Core enzyme binding is more transient (t1/2 ≤
1min) 6-7
 Filter Binding
 Filter binding is used to measure DNA-
protein interaction and based on the
fact that double-stranded DNA will not
bind by itself to a filter, but a protein-
DNA complex will.
Double-stranded DNA can be labeled and
mixed with protein
Assay protein-DNA binding by measuring
the amount of label retained on the filter

5-8
 Temperature and RNA Polymerase
Binding (flip expt)
 3H-labeled T7 phage DNA
with holoenzyme - then they
added excess cold DNA –
Passed reactions thru
nitrocellulose filter.
 As the temperature is
lowered, the binding of RNA
polymerase (holoenzyme) to
DNA decreases dramatically.
 Higher temperatures
promote DNA melting and
encourage holoenzyme
binding.
6-9
 RNA Polymerase Binding
Hinkle and Chamberlin proposed:
 RNA polymerase holoenzyme binds DNA
loosely at first.
 Binds at promoter initially
 Scans along the DNA until it finds a promoter
 Complex with holoenzyme loosely bound at
the promoter is a 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.
6-10
 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.

6-11
 Summary

 The -factor allows initiation of

transcription by causing the RNA polymerase
holoenzyme to bind tightly to a promoter.
 This tight binding depends on local melting
of the DNA to form an open promoter
complex and is stimulated by .
 The -factor can therefore select which
genes will be transcribed, thus its name of
specificity factor.

6-12
 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, a.k.a “Pribnow box”.
 Another short sequence centered 35 bp
upstream is known as the -35 box.
 Comparison of thousands of promoters has
produced a consensus sequence (or most
common sequence) for each of these boxes.

6-13
 Promoter Strength
 Consensus sequences:
 -10 box sequence approximates TAtAaT
 -35 box sequence approximates TTGACa
 Mutations that weaken promoter binding:
 Down mutations
 Increase deviation from the consensus sequence
 Mutations that strengthen promoter binding:
– Up mutations
– Decrease deviation from the consensus sequence

6-14
 UP Element
 The UP element is upstream of the core
promoter, stimulating transcription by a factor
of 30 (e.g. rrnB P1 gene).
 This promoter is associated with 3 “Fis” sites
(-60 and -150), which are binding sites for the
transcription-activator protein Fis, not for the
polymerase itself, and are called enhancers.

6-15
 The rrnB P1 Promoter

 Transcription from the rrn promoters responds

 Positively to increased concentration of iNTP,
which stabilize open promoter complex
 Negatively to the alarmone (guanosine 5’-
diphosphate 3’-diphosphate), ppGpp, produced
by the ribosome-associated protein RelA, which
senses the lack of amino acids
 Is Regulated by DskA, which binds RNA
polymerase and reduces the lifetime of open
promoter complex
6-16
 6.3 Transcription Initiation
 Transcription initiation 1. RNA Pol + lac UV5 promoter
was assumed to end as 2. + Heparin and 32P-ATP
3. Gel electrophoresis
RNA polymerase formed
1st phosphodiester bond.
 Carpousis and Gralla
found that very small
oligonucleotides (2-6 nt
long) are made without
RNA polymerase leaving
the promoter.
 Abortive transcripts
such as these have been
found up to 9 or 10 nt.
6-17
 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 (initial
transcribing complex).
 Promoter clearance –
transcript becomes long
enough to form a stable
hybrid with template.
6-18
 Sigma Stimulates Initiation of
Transcription but not Elongation
 In this first experiment of
Travers and Burgess, σ
appears to stimulate both Elongation

initiation and elongation on Initiation

T4 DNA.
 Or stimulating initiation by
σ provides more initiated
chains for core polymerase
to elongate.
 Further experiments by
the same group proved that
σ does not stimulate
elongation.
6-19
 Reuse of σ
- Rifampicin

+ Rifampicin

Low ionic strength

 During initiation σ can be recycled for additional use

with a new core polymerase.
 The holoenzyme can release σ, which is then free to
associate with new core enzyme.
 Travers and Burgess dubbed this the “σ cycle”, with
an obligate release mechanism.
6-20
Fluorescence Resonance Energy Transfer
 The -factor changes its relationship to the core
polymerase during elongation (release might occur
during elongation at +16/+17).
 σ-factor may not dissociate from the core but
actually shift position and become more loosely
bound to core.
 To answer this question Fluorescence Resonance
Energy Transfer (FRET) was used as it relies on two
fluorescent molecules that are close enough
together to engage in transfer of resonance energy.
 FRET allows the position of σ relative to a site on
the DNA to be measured without using separation
techniques that might displace σ from the core
enzyme.
6-21
FRET Assay for σ Movement Relative to
DNA

 The FRET efficiency increase

suggests that 100% of the
complexes retain .
6-22
 Models for the -Cycle
 The obligate release version of the -cycle
model arose from experiments performed by
Travers and Burgess that proposed the
dissociation of  from core as polymerase
undergoes promoter clearance and switches
from initiation to elongation mode.
 The stochastic release model proposes that
 is indeed released from the core
polymerase but that there is no discrete point
of release during transcription and that the
release occurs at random - a preponderance
of evidence favors this model.
6-23
Local DNA Melting at the Promoter
 From the number of RNA polymerase
holoenzymes bound to phage T7 DNA (frgt
with 3 promoters), it was calculated by
hyperchromic shift that each polymerase
caused a separation of about 10 bp.
 In another experiment (end-labeling + DMS +
S1 nuclease), the length of the melted region
was found to be 12 bp (see next slide).
 Later, size of the DNA transcription bubble in
complexes where transcription was active was
found to be 17-18 bp (see next to next slide).

6-24
 Promoter Clearance
 RNA polymerases have evolved to
recognize and bind strongly to
promoters.
 This poses a challenge when it comes
time for promoter clearance as those
strong bonds must be broken in order
for polymerase to leave the promoter
and enter the elongation phase.

6-25
 Promoter Clearance Cont.
 Several hypotheses have been proposed.
 The polymerase cannot move enough
downstream to make a 10 nt transcript without
doing one of three things:
- transient excursion: moving briefly
downstream and then snapping back to the
starting position
- inchworming: stretching itself by leaving its
trailing edge in place while moving its leading
edge downstream
- scrunching: compressing the DNA without
moving itself
6-26
 Structure and Function of σ
 Genes encoding a variety of σ-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.

6-27
Homologous Regions in Bacterial 
Factors

6-28
 E. coli σ70
 Four regions of high sequence similarity are
indicated.
 Specific areas that recognize the core
promoter elements are the -10 box and –35
box.

6-29
 Region 1
 Role of region 1 appears to be in preventing σ
from binding to DNA by itself.
 This is important as  binding to promoters
could inhibit holoenzyme binding and thereby
inhibit transcription.
Region 2
 This region is the most highly conserved.
 There are four subregions: 2.1 to 2.4
 2.4 recognizes the promoter’s -10 box.
 The 2.4 region appears to be an -helix.

6-30
 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

6-31
 Analysis of binding between σ region 4.2 and
the -35 box, as well as in the presence of β’
fragment
Nitrocellulose
binding of
labeled ptac DNA +
GST-4.2 protein

SDS-PAGE on
UV cross-linked
complexes
(binding to the -10
box)

6-32
 Summary
 Comparison of different σ 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 σ-factor by itself cannot bind to DNA,
but DNA interaction with core (β’) unmasks a
DNA-binding region of σ.
 Region between amino acids 262 and 309 of
β’ stimulates σ binding to the nontemplate
strand in the -10 region of the promoter.
6-33
 Role of -Subunit in UP Element
Recognition

 RNA polymerase itself can recognize an

upstream promoter element, UP element.
 While σ-factor recognizes the core
promoter elements, what recognizes the
UP element?
 It appears to be the -subunit of the
core polymerase.

6-34
 Role of α-Subunit in UP Element Recognition Cont.
Gourse and
Co-workers
In vitro
transcription

6-35
 Modeling the Function of the C-
Terminal Domain
 RNA polymerase binds to a
core promoter via its σ-
factor, no help from C-
terminal domain of α-
subunit.
 Binds to a promoter with
an UP element using σ plus
the -subunit C-terminal
domains (CTD).
 Results in very strong
interaction between
polymerase and promoter.
 This produces a high level
of transcription.
6-36
 6.4 Elongation
 After transcription initiation is
accomplished, core polymerase
continues to elongate the RNA.
 Nucleotides are added sequentially,
one after another in the process of
elongation.

6-37
 Structure of the Elongation
Complex
 This section will examine how well
predictions have been born out by
structural studies.
 How does the polymerase deal with
problems of unwinding and rewinding
templates?
 How does it move along the helical
template without twisting RNA product
around the template?

6-38
 RNA-DNA Hybrid
 The area of RNA-DNA hybridization within the E.
coli elongation complex has been controversial with
estimates ranging from 3-12 bp.
 Nudler and Goldfarb used a transcript walking
technique combined with RNA-DNA cross-linking to
show that this hybrid is 8-9bp long.
 They His-tagged RNA polymerase by introducing 6
histidines in the  subunit.
 Once immobilized on nickel resin, they can control
reaction conditions by controlling the nucleotide
added to the reaction.
 Walk the polymerase to a specific position where the

6-39
 RNA-DNA Hybrid Cont.
 first UTP is required, then wash and add a second
subset of nucleotides to walk polymerase further
downstream.
 Using a UMP derivative (U), Nudler and Goldfarb
Incorporated U at either position 21 or 45, and showed
that the RNA-DNA hybrid exists in region from -2 to -8.

6-40
Structure of the Core Polymerase

 X-ray crystallography on the Thermus

aquaticus RNA polymerase core reveals
an enzyme shaped like a crab claw.
 It appears designed to grasp the DNA.
 A channel through the enzyme includes
the catalytic center.
Mg2+ ion coordinated by 3 Asp residues
Rifampicin-binding site

6-41
 Structure of the Core Polymerase Cont.
 3.3Å structure shows an
open crab claw with a
channel 27Å wide.
 Half the claw is composed
of , and the other half is
made of ’.
 The two  subunits lie at
the hinge of the claw, with
I associated with  and II
associated with ’.
  subunit is at the bottom
in an interaction with the C-
terminus of ’.
6-42
 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 rewind
behind it.
 Strain introduced into the
template DNA ahead of the
transcription bubble is relaxed by
topoisomerases.
6-43
 Pausing and Proofreading
 RNA polymerase frequently pauses, or even
backtracks, during elongation.
 Pausing allows ribosomes to keep pace with
the RNA polymerase, and it is the first step
in termination.
 Backtracking aids proofreading by extruding
the 3’-end of the RNA out of the polymerase,
where misincorporated nucleotides can be
removed by an inherent nuclease activity of
the polymerase, stimulated by auxiliary
factors GreA (2-3nt) and GreB (up to 18nt).
6-44
 6.5 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 rho (, these are rho or -dependent
terminators)

6-45
 Rho-Independent Termination

 Intrinsic or rho-independent
termination depends on terminators of
2 elements:
Inverted repeats followed immediately by
T-rich region in the nontemplate strand of
the gene
 An inverted repeat predisposes a
transcript to form a hairpin structure
due to complementary base pairing
between the inverted repeat sequences.

6-46
 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

6-47
 Structure of an Intrinsic Terminator
 Attenuator contains a DNA sequence that causes
premature termination of transcription.
 The E. coli trp attenuator was used to show:
 Inverted repeat allows a hairpin to form at transcript end
 String of T’s in nontemplate strand result in weak rU-dA
base pairs holding the transcript to the template strand
 Mutations in either the GC-rich hairpin or the T-rich
sequence abolish attenuator function
AU
GC
CG
CG
CG
GC
CG A
CG
U U
A A
6-48
 Model of Intrinsic Termination
Bacterial terminators act by:
 Base-pairing of something to
the transcript to destabilize
RNA-DNA hybrid
 Causes hairpin to form
 This causes transcription to
pause
 a string of U’s incorporated
just downstream of hairpin to
destabilize the hybrid and the
RNA falls off the DNA
template
6-49
 Rho-Dependent Termination

 Jeffery Roberts discovered that Rho

caused depression of the ability of RNA
polymerase to transcribe phage DNAs in
vitro.
 This depression was due to termination
of transcription.
 After termination, polymerase must
reinitiate to begin transcribing again.

6-50
 Rho Affects Chain Elongation
 There is little effect of
rho on transcription
initiation, if anything it is
increased.
 The effect of rho on
total RNA synthesis is a
significant decrease.
 This is consistent with
action of rho to
terminate transcription
forcing time-consuming
reinitiation of short
transcripts. 6-51
 Rho Causes Production of Shorter
Transcripts
 Synthesis of much smaller
RNAs occurs in the presence
of rho compared to those
made in its absence.
 To ensure that this due to
rho itself and not to RNase
activity of rho, RNA was
transcribed without rho and
then incubated in the
presence of rho.
 There was no loss of
transcript size, so no RNase
activity in rho.

6-52
 Rho Releases Transcripts from the DNA
Template
 Compare the sedimentation of
transcripts made in presence
and absence of rho.
 Without rho, transcripts
cosedimented with the DNA
template
 With rho present in the
incubation, transcripts sedimented
more slowly – they were not
associated with the DNA template
 It appears that rho releases
RNA transcripts from the DNA
template.
6-53
 Mechanism of Rho
 No string of T’s in the rho-dependent
terminator, just inverted repeat to
hairpin.
 rho binds RNA polymerase when
transcript is still very short and moves
along with it as it elongates.
 When RNA grows longer and contains
rho loading site (rut), it binds to rho.
 As transcription progresses, rho feeds
RNA product thru hole.
 Once polymerase pauses, rho tighten
the RNA loop and inhibits transcription.
 Rho releases transcript from the DNA-
polymerase complex by unwinding the
RNA-DNA hybrid.
6-54
 Summary
 Using the trp attenuator as a model rho-
independent terminator revealed two important
features:
1 - an inverted repeat that allows a hairpin to form
at the end of the transcript
2 - a string of T’s in the nontemplate strand that
results in a string of weak rU-dA base pairs holding
the transcript to the template strand
 Rho-dependent terminators consist of an inverted
repeat, which can cause a hairpin to form in the
transcript but no string of T’s.
6-55
Student Expected Learning Outcomes
1. Discuss Structure and function of bacterial RNA
polymerase.
2. Identify the different bacterial core promoter
elements and discuss the important role played by
sigma factor in their recognition.
3. Explain how RNA polymerase is recruited to the
promoter region, and identify the different stages of
transcriptional regulation.
4. Understand how sigma factor contributes to
transcriptional initiation, and how transcriptional
elongation proceeds followed by either rho-dependent
or rho-independent termination.

6-56