Transcription in prokaryotes
Part 1
RNA Polymerase and
Promoter Structure
Steps of transcription
1.
Steps of transcription
Initiation:
RNA polymerase recognizes gene promoter and binds to it. Promoter sequence is
melted and RNA polymerase begins synthesis of RNA chain.
2.
Elongation:
RNA polymerase moves along DNA template melting DNA sequence and
extending RNA chain in 5’ → 3’ direction. Only one strand is transcribed
(asymmetrical transcription). DNA melting is limited to RNA polymerase
binding site and is transient, once ‘reading’ is completed DNA restores its original
conformation.

3.
compare to DNA replication, where both strands are copied
simultaneously and two original strands are separated permanently.
Termination:
RNA polymerase reaches the terminator; RNA product dissociates from RNA
polymerase and DNA template; transcription stops.
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RNA polymerase/promoter binding
Prokaryotic promoters
• All prokaryotic promoters have two common regions (core
promoter elements) centered at -10 and -35bp upstream of the
transcription start site.
Holoenzyme searches for a promoter
• These consensus sequences are TATAAT (at -10bp) and
TTGACA (at -35bp)
Once promoter was found the closed
promoter complex is formed
Enzyme binding tightens, local DNA
region melts and open promoter
complex is formed
• Mutation that destroy matches in consensus sequences are
called down mutations (weakening promoter); mutation that make
promoter sequences more like consensus sequences are called
up mutations (promoter becomes stronger)
 The more closely core promoter elements resemble the
consensus sequences, the stronger promoter will be.
Prokaryotic promoters
Prokaryotic promoters
• Presence of UP elements located farther upstream from core
elements creates very strong promoters
• UP elements represent the true promoter elements; they are
recognized by RNA polymerase itself and can stimulate
transcription by a factor of 30 (UP element from rrnB P1 promoter)
 Capital letters – bases found in these positions in more
than 50% of promoters.
 Small letters – bases found in 50% or fewer of
promoters studied.
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Prokaryotic promoters
The rrnB P1 promoter
•
DNA sites that do not bind to RNA polymerase itself, but
can stimulate transcription through the binding to the transcriptionactivator proteins are called enhancers. Enhancers represent a
class of not classical promoter elements (Fis protein-activator binds
Fis sites in rrnB P1 promoter).
• rrnB P1 promoter can be also regulated by a number of small
molecules that stabilize (i.e. iNTP) or destabilize (i.e. alarmone)
open promoter complex. The promoter’s ability to respond to this
type of regulation is dependent on an open promoter lifetime,
which is under the negative control of DskA protein.
Burgess and Travers experiment:
RNA polymerase structure
E. coli RNA polymerase subunits in SDS-PAGE
E. coli RNA polymerase subunits:
β – 150 kD
β’ – 160 kD
σ – 70 kD
α – 40 kD
 RNA polymerase holoenzyme = β, β’, σ, α2
 RNA polymerase core = β, β’, α2
•
RNA pol holoenzyme was subjected to anion exchange chromatography
using phosphocellulose resin; three peaks were detected.
•
Three protein peaks were analyzed in SDS-PAGE
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Transcription of nicked DNA: Why?
Why core is still capable of transcribing nicked DNA
template, but not intact???
 Separation of σ - factor from RNA holoenzyme dramatically
decreased enzyme activity
 Adding σ - factor back rescued the enzyme activity

Nicks and gaps provide ideal, yet unspecific initiation sites
for RNA polymerase, even for core

Few nicks and gaps occurred on intact T4 served as
artificial initiation sites; therefore, DNA was still weakly
transcribed
 Presence of σ- factor allows recognition of specific
transcription start sites on T4 DNA
 Transcription of nicked DNA represents a laboratory
artifact
Experiment of Bautz et al :
Role of σ factor
DNA
Bautz et al :
RNA
In vivo
transcriptio
n
• Holoenzyme transcribes only certain T4 genes
(immediate early genes)
DNA
• Core enzyme shows no such specificity and transcribes
from both DNA strands
RNA
DNA
RNA
RNA
Asymmetric
In vitro
transcription
(holoenzy me)
Symmetric In vitro transcription (core)
RNA
RNA
RNase sensitive
dsRNA
RNase resistant
RNA
RNA
RNase sensitive
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Role of σ factor
Experiment of Bautz et al :
• RNase used in experiment could degrade single but not
double stranded RNA molecules
Bautz et al.
• About 30% of the labeled RNA made by core enzyme in
vitro became insensitive to RNase after hybridization with
authentic T4 DNA
 A core enzyme has basic RNA synthesis capabilities but
lacks specificity
Conclusions :
 Presence of σ - factor directs transcription of
holoenzyme to specific genes
• RNA produced in vitro by holoenzyme did not hybridized
with authentic T4 DNA and remained RNase sensitive
Hinkle and Chamberlin experiment
Hinkle and Chamberlin experiment
Set-up:
Results:
• 3H-labeled T7 DNA was allowed to bind to holoenzyme
and to core.
• Holoenzyme was
dissociating much slower
than the core.
• Unlabeled DNA was added in access – polymerase
molecules that dissociate from labeled DNA would likely bind
to unlabeled DNA.
• Mixtures were filtered through nitrocellulose at various
times allowing to monitor dynamics of binding and dissociation.
• When the last polymerase dissociates from the labeled
DNA, the DNA will be no longer bound to the filter, which loses
radioactivity.
• Thus – holoenzyme
binds tightly.
• Thus - σ promotes tight
binding
 σ stimulates tight binding between RNA polymerase and
promoter
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Hinkle and Chamberlin: Conclusions
• Chamberlin et al. showed that tight complexes between
T7 DNA and holoenzyme could initiate transcription
immediately upon addition of nucleotides.
•
Holoenzyme binds loosely to DNA at first.
•
It either binds to a promoter or scans for it.
• Latter reinforces the conclusion that binding occurs at
promoters
•
 Titration of tight binding sites yielded only 8 sites
that corresponded to the number of early promoters.
Complex of holoenzyme bound loosely to DNA – a
closed promoter complex – DNA remains doublestranded
•
 Number of loose sites was more than 1300. All of
them were nonspecific.
Holoenzyme can melt a short region of the DNA at
promoter – an open promoter complex is formed.
•
Conversion from closed to open promoter complex
requires σ-factor.
•
σ factor selects promoter to which polymerase can bind
tightly, therefore allowing transcription of genes driven
by this promoter
NB: The inability of core to bind to the tight (promoter)
binding sites accounts for its inability to transcribe
DNA specifically, which requires binding at promoters.
Polymerase
/promoter
binding
Interaction of RNA
polymerase holoenzyme and
promoter
σ Binds core with closed channel to form
open complex
Holoenzyme binds loosely
Holoenzyme grasps DNA tightly
σ dissociates, leaving core clamp around
DNA
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Reading:
R. Weaver, Molecular Biology, 4th ed.
Chapter 6: pages 127-132.
Carpousis and Gralla experiments:
• E.coli RNA polymerase was incubated with DNA bearing
promoter (lacUV5) + heparin (it competes with DNA for
binding to polymerase).
• Heparin prevented re-association of polymerase released
at the end of transcription cycle and DNA.
• Also they included labeled ATP to label RNA products.
Outcome:
• Several very small oligonucleotides (2-6 nt long) were
found, which sequences matched the sequence of the
beginning of the expected transcript.
• Many oligos were produced per polymerase.
• Polymerase was making small oligos (abortive transcripts)
without leaving promoter.
Part 2:
Transcription Initiation
Current view on initiation:
1) Closed promoter complex formation
2) Conversion of closed complex into open promoter
complex
3) Polymerizing the first few nucleotides (up to 10) while
polymerase remains at the promoter
4) Promoter clearance, in which transcript becomes long
enough to form a stable hybrid with a template strand.
This helps to stabilize the transcription complex.
Polymerase changes to its elongation conformation,
looses its σ-factor and moves away from the promoter.
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Functions of σ factor
• Selects genes for transcription causing tight binding
between RNA pol and promoter
•
The tight binding depends on local melting of the DNA.
• σ-factor can dissociate from core after initiating polpromoter binding
 σ-factor stimulates transcription initiation.
 σ-factor does not really accelerate the rate of chain
growth.
 Initiation is a rate-limiting step in transcription (it takes
longer to get new RNA chain started than to elongate the
existing one).
σ-Factor cycle
Local DNA melting at the promoter
•
Hsieh and Wang, 1978: binding RNA pol to the DNA
fragment containing three T7 early promoters and
measuring hyperchromic shift – RNA pol melts about 10bp
•
Siebenlist, 1979: RNA pol melts about 12bp
 RNA pol binds to promoter, causing local melting. When pol
moves elongating RNA, sigma factor dissociates, and can be
reused by a new core pol, initiating another RNA chain.
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Experiment of Ulrich Siebenlist
Set-up:
Experiment of Ulrich Siebenlist
Set-up:
1. End-labeled promoter DNA
2. Added RNA pol to form open promoter complex (contains
melted DNA). When strands separate the N1 of adenine
becomes exposed and susceptible to certain chemical agents.
3. The exposed A were methylated with DMS preventing future
pairing with thymine.
4. Once RNA pol was removed the melted region closed up
again; base-pairing between T and CH3 -A was disturbed –
regions of ssDNA were left.
5. DNA was treated with S1 nuclease, that cuts ssDNA – the
enzyme should have cut wherever A has been in melted
region and had become methylated (here DNA remained single
stranded).
6. This should have produced a series of end-labeled products,
each one terminated at A.
7. Electrophoresis could show the length of products. These
lengths and the knowledge of exact position of labeled end
allowed to calculate the position of melted region.
Experiment of Ulrich Siebenlist
Further data:
Gamper and Hearst: demonstrated that polymerase binds
to the promoter, melting 17±1 bp of DNA to form
transcription bubble, and a bubble of this size moves
with polymerase as it transcribes DNA.
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Extent of polymerase binding to promoters
Extent of polymerase binding to promoters
Gilbert et al:
1. Labeled DNA containing promoter at one of its 5’ ends
2. Bound RNA pol to promoter
3. Treated complex with exonuclease III, which degrades
DNA base by base from 3’ ends.
4. This left a labeled DNA stand whose upstream end has
been chewed off to the point where polymerase blocked
further degradation.
5. Released protected DNA, denatured, electrophoresed.
6. Observed a major band extending to position –44 from at
least +2.
The structure of sigma factor
Conclusion:
Polymerase extends from position –44 to +2 in
an open promoter complex.
The structure of sigma factor
• Genes encoding sigma factors have been cloned and
sequenced.
•
E.coli has a primary sigma factor σ 70 (named after its
molecular mass).
• There are alternative ones, transcribing specialized genes
(heat shock, sporulation, etc).
•
Many factors were analyzed and similarities recorded.
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The structure of sigma factor
•
Regions of primary structure in E. coli σ70
σ70 of E. coli has 245aa, that allows it to loosen binding
between polymerase and non-promoter regions.
•
σ43 of B. subtilis can not do this. It required extra factor - δ.
-10 box recognition
-35 box recognition
 The E. coli σ factor can loosen nonspecific interactions between
polymerase and DNA and thus enhance the specificity of the
polymerase-promoter interactions.
 The B. subtilis σ factor lacks this activity. It needs the delta-factor.
Regions:
Regions:
Region 1:
It is found only in primary σ factors.
Region 2:
It is highly conserved.
Role:
Role:
• It prevents sigma from binding by itself to DNA in the
absence of core. The latter is important, since binding of
sigma to promoters may inhibit holoenzyme binding and
transcription.
• 2.1 – involved in binding to RNA pol core. It is necessary
and sufficient for it. It has hydrophobic patch.
• 2.4 – responsible for recognition of -10 box. This region
contains an amino acid sequence that can form α helix – a
favorite DNA binding motif.
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Regions:
Regions:
Region 4:
Region 3:
It is absent in many sigma factors.
It is found in almost all sigma factors.
Role:
Role:
•
It can form helix-turn-helix DNA-binding domain
•
It is involved in both core and DNA binding
• 4.2 – contains a helix-turn-helix DNA-binding domain
and plays a key role in promoter recognition (binding to the 35 box)
NB: Both subregions 2.4 and 4.2 are capable of binding promoters
on their own, but other domains interfere with this binding.
The role of α-subunit
The role of α-subunit
Gourse et al:
• It has independently folded C-terminal domain that can
recognize and bind to a promoter UP element.
 Some of E. coli strains with mutations in the α subunit are
incapable of responding to the UP element – they give no
more transcription from promoters with elements than from
those without.
• This allows very tight binding between polymerase and
promoter.
 DNase footprinting experiments with DNA containing rrnB
P1 promoter and either wild type or mutant polymerase
indicated that the α-subunit C-terminal domain is required for
UP and polymerase interactions.
 Limited proteolysis indicated that the α-subunit N-terminal
and C-terminal domains fold independently to form two
domains that are tethered together by a flexible linker.
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Model of the function of the C-terminal domain (CTD) of
the polymerase α-subunit
Reading:
R. Weaver, Molecular Biology, 4th ed.
Chapter 6: pages 132-146.
W/o details of figures 6.17; 6.22-6.25
 In a promoter with UP the CTD is used to contact UP element.
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