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Chapter 6.

The Mechanism of Transcription


in Prokaryotes
E. coli RNA Polymerase

160 kD

150 kD

70 kD

40 kD

RNA polymerase holoenzyme: β’, β, σ, α 2


RNA core polymerase: β’, β, α 2
E. coli RNA Polymerase
Sigma (σ) as a Specificity Factor

- Holoenzyme transcribes only a certain class of T4 genes


(“immediate early genes”).

- Core enzyme shows no such specificity.


Sigma (σ) as a Specificity Factor

al transcription can be divided into three phases.


Sigma (σ) as a Specificity Factor
How did Bautz and colleagues show that σ restored
specificity to the nonspecific core?

 “Hybridization competition”
Sigma (σ) as a Specificity Factor
Sigma (σ) as a Specificity Factor

Green: 1min
Red: 5 min
Blue: 17 min after infection
with T4 phage
The Core Enzyme Transcribes
Both DNA Strands
ting for asymmetric or symmetric transcription in vitro
g hybridization and checking subsequent RNase resist

Core enzyme

Holoenzyme
Sigma (σ) as a Specificity Factor
hy was core RNA polymerase still capable of transcrib
nicked DNA, but not intact DNA?
Sigma (σ) as a Specificity Factor
hy was core RNA polymerase still capable of transcrib
nicked DNA, but not intact DNA?

Nicks and gaps in DNA provide ideal initiation sites for


RNA polymerase, even core polymerase.
Promoters

- Specific RNA polymerase binding sites are called


“promoters”.

− σ confers specificity to the RNA polymerase so that


it initiates transcription at specific promoters.
Binding of RNA Polymerase to Promoters
Nitrocellulose filter-binding studies
performed by Hinkle and Chamberlin
Binding of RNA Polymerase to Promoters
Nitrocellulose filter-binding studies
performed by Hinkle and Chamberlin
Binding of RNA Polymerase to Promoters
Nitrocellulose filter-binding studies
performed by Hinkle and Chamberlin

+ An excess of
unlabeled DNA
Binding of RNA Polymerase to Promoters
Nitrocellulose filter-binding studies
performed by Hinkle and Chamberlin

+ An excess of
unlabeled DNA
Binding of RNA Polymerase to Promoters

- The polymerase holoenzyme


binds much more tightly to
the T7 DNA than does the
core enzyme.

- Thus, the σ-factor can pro-


mote tight binding, at least
to certain DNA sites
(promoters).
Enhancement of Tight Binding at
Elevated Temperature

- Tight binding involves


local melting of the DNA.
RNA Polymerase/Promoter Binding
A model for RNA polymerase/promoter binding
proposed by Hinkle and Chamberlin
Promoter Structure
Promoter Structure

Mark Ptashne David Pribnow


Promoter Structure

Consensus sequences

Down mutations: Mutations that destroy matches with


he consensus sequences  make the promoter weaker

Up mutations: Mutations that make the promoter sequence


more like the consensus sequences  make the promoter
stronger
Promoter Structure

Spacing is also important.

“Core promoter elements”: -10 and -35 boxes


The rrnB P1 Promoter

UP Core
element promoter

The UP element is recognized by RNA polymerase, so


it is a promoter element.
The rrnB P1 Promoter

UP Core
Fis sites element promoter

Fis sites are recognized by the transcription-activator


protein Fis.  Transcription-activating DNA elements
(Enhancers)
Transcription Initiation
Agamemmon Carpousis and Jay Gralla reported that
transcription initiation is complex.

E. coli RNA polymerase + DNA bearing lac UV promoter


Labeling of the RNA products
with [32P]ATP

Addition of heparin to the mixture


(A negatively charged polysaccharide that competes with
DNA in binding tightly to free RNA polymerase)
Transcription Initiation
- They found several very small
oligonucleotides, ranging in size
from dimers to hexamers.

- Many oligonucleotides/polymerase

- “Abortive transcription” by RNA


polymerase, without leaving the
promoter

- Abortive transcripts up to 9 or 10 nt
in size
Transcription Initiation

“Promoter clearance”:
The transcript becomes long
enough to form a stable hybrid
with the template strand

The polymerase changes to


its elongation conformation,
losing its σ-factor and moves
away from the promoter.
σ Stimulates Transcription Initiation
Experiments performed by Travers and Burgess

(pyrophosphate)

Phosphodiester bond formation in RNA synthesis


σ Stimulates Transcription Initiation
Experiments performed by Travers and Burgess

Incubation of core polymerase in the presence of increasing


amounts of σ in two separate sets of reactions

[14C]ATP: [γ-32P]ATP or [γ-32P]GTP:


Incorporated throughout Incorporated only into the
the RNA first position of the RNA
 Measurement of both
initiation and elongation
σ Stimulates Transcription Initiation
Experiments performed by Travers and Burgess

σ seems to stimulate both


initiation and elongation.
σ Stimulates Transcription Initiation
Experiments performed by Travers and Burgess

σ seems to stimulate both


initiation and elongation.

However, initiation is the


rate-limiting step in transcr-
iption. Thus, it appears that
σ stimulates elongation by
stimulating initiation.
σ Stimulates Transcription Initiation,
but not Elongation
Experiments performed by Travers and Burgess
 Examination of the effect of σ on the length
of the constant number of RNA chains
σ Stimulates Transcription Initiation,
but not Elongation
Experiments performed by Travers and Burgess
 Examination of the effect of σ on the length
of the constant number of RNA chains

Allowed a certain amount of initiation to occur

Treatment with rifampicin, which blocks prokaryotic


transcription initiation, but not elongation

Measurement of the length of RNAs made in the presence


or absence of σ
σ Stimulates Transcription Initiation,
but not Elongation
Experiments performed by Travers and Burgess
 Examination of the effect of σ on the length
of the constant number of RNA chains

Allowed a certain amount of initiation to occur

Treatment with rifampicin, which blocks prokaryotic


transcription initiation, but not elongation

Measurement of the length of RNAs made in the presence


or absence of σ
“No difference in the lengths of the RNAs”
Reuse of σ
Experiments performed by Travers and Burgess
Transcription reaction at low ionic strength, which prevents
core polymerase from dissociating from the DNA template
at the end of a gene
Reuse of σ
Experiments performed by Travers and Burgess

Addition of new, rifampicin-


resistant core polymerase in
the presence (green) or absence
(blue) of rifampicin

Transcription begins anew,


meaning that the new core
associates with σ that had been
released from the original
holoenzyme
Reuse of σ
Experiments performed by Travers and Burgess

Addition of new, rifampicin-


resistant core polymerase in
the presence (green) or absence
(blue) of rifampicin

Transcription begins anew,


meaning that the new core
associates with σ that had been
released from the original
holoenzyme
“The core, but not σ, determines
rifampicin resistance or sensitivity”
The σ Cycle Hypothesis
Sigma May Not Dissociate from Core
During Elongation

1. Jeffrey Roberts and colleagues (1996):


σ is involved in pausing at position +16/+17 downstream
of the late promoter PR’ in λ phage.
 σ is still attached to core polymerase at the position,
well after promoter clearance has occurred.

2. Richard Ebright and coworkers (2001):


All of the evidence favoring the σ cycle model relies on
harsh separation techniques, which could strip σ off of core
if σ is weakly bound to core during elongation
Sigma May Not Dissociate from Core
During Elongation
Ebright and coworkers:
“Fluorescence Resonance Energy Transfer (FRET)”
 Two fluorescent molecules close to each other engage
in transfer of resonance energy, and the efficiency of
the energy transfer (FRET efficiency) decreases rapidly
as the two molecules move apart.
Sigma May Not Dissociate from Core
During Elongation
Ebright and coworkers:
Measurement of FRET with fluorescent molecules
(fluorescence probes) on both σ and DNA

• The probe on σ as the fluorescence donor


• The probe on the DNA as the fluorescence acceptor
Sigma May Not Dissociate from Core
During Elongation
Ebright and coworkers:
Measurement of FRET with fluorescent molecules
(fluorescence probes) on both σ and DNA

• The probe on σ as the fluorescence donor


• The probe on the DNA as the fluorescence acceptor

- Trailing-edge FRET: The probe on the DNA is at the 5’,


or upstream end  Observation of the drop in FRET as
the polymerase moves away from the promoter

- Leading-edge FRET: The probe on the DNA is at the 3’,


or downstream end  Observation of the increase in FRET
as the polymerase moves toward the downstream end
Rationale of FRET Assay for σ Movement
Relative to DNA

: Fluorescence
acceptor

: Fluorescence
donor
FRET Analysis of σ-core Association After
Promoter Clearance

: FRET efficiency (E)


of open promoter
complex
: FRET efficiency (E)
after 5 and 10 min
FRET Analysis of σ-core Association After
Promoter Clearance

RET did increase, supporting that σ


emains with the core after promoter
learance
σ-core Association After Promoter Clearance
Bar-Nahum and Nudler (2001):

Purification of elongation complexes stalled at position +32


(EC32) from stationary cells or from exponentially growing
cells
σ-core Association After Promoter Clearance
Bar-Nahum and Nudler (2001):

Purification of elongation complexes stalled at position +32


(EC32) from stationary cells or from exponentially growing
cells
Local DNA Melting at the Promoter

Tao-shih Hsieh and James Wang (1978):


Direct evidence for local DNA melting

E. coli RNA polymerase + T7 early promoters

Measurement of the hyperchromic shift, which is not


only indicative of DNA strand separation but also its
magnitude is directly related to the number of bps that
are opened.
Local DNA Melting at the Promoter

Tao-shih Hsieh and James Wang (1978):


Direct evidence for local DNA melting

E. coli RNA polymerase + T7 early promoters

Measurement of the hyperchromic shift, which is not


only indicative of DNA strand separation but also its
magnitude is directly related to the number of bps that
are opened.

Each polymerase causes a separation of ~10 bp.


Local DNA Melting at the Promoter
Ulrich Siebenlist’s identification of the base pairs that
RNA polymerase melted in a T7 early promoter

Dimethyl sulfate
(DMS)

End-labeling of the Closing up again of


promoter DNA the melted region by
removing the RNA
Formation of an open polymerase
promoter complex by
adding RNA polymerase
Local DNA Melting at the Promoter

* S1 nuclease specifically cuts


single-stranded DNA.
Local DNA Melting at the Promoter

- Melted region of ~ 12 bp
- The melted region is just at the place
where RNA polymerase begins transcribing.
Local DNA Melting at the Promoter
Howard Gamper and John Hearst (1982):

Estimation of the number of bps melted by polymerases,


not only in binary complexes, but also in actively transcribing
complexes that also contained RNA (ternary complexes)

SV40 DNA with one promoter site recognized by the E. coli


RNA polymerase + RNA polymerase

At either 5oC or 37oC


In the absence of nucleotides to form binary complexes
Or in the presence of nucleotides to form ternary complexes
Local DNA Melting at the Promoter

Relaxing of any supercoils

The underwinding introduces strain into


the closed circle that is relieved by forming
supercoils.

The higher the superhelical content, the


greater the double helix unwinding that
has been caused by the polymerase
Local DNA Melting at the Promoter

Measurement of the superhelical


content of a DNA by gel electro-
phoresis:

The more superhelical turns a


DNA contains, the faster it will
migrate in a gel.
Local DNA Melting at the Promoter
~1.6 superhelical turns/
one polymerase

Each polymerase unwinds 1.6 turns


of the DNA double helix

If a double helical turn contains


10.6 bp, then each polymerase
melts 17 bp (1.6 X 10.6)

“Transcription bubble”:
Moves with the polymerase
as it transcribes the DNA
Structure and Function of σ

A variety of σ-factors from various bacteria

1) Primary σ-factors (σA) that transcribe vegetative genes,


which are required for everyday growth
 E. coli : σ70, B. subtilis : σ43

2) Alternative σ-factors that transcribe specialized genes


(heat shock genes and sporulation genes etc.)
Homologous Regions in Various σ Factors
Structure and Function of σ

• Region 1: - Present only in the primary σ’s


- Plays a role to prevent the whole σ from binding
by itself to DNA in the absence of core polyme-
rase

2. Region 2: - Present in all σ-factors


- Subdivided into four parts, 2.1-2.4

- Subregion 2.4 is responsible for a crucial σ


activity, recognition of the promoter’s -10 box.
Structure and Function of σ

3. Region 3: - Involved in both core and DNA binding

4. Region 4: - Subdivided into subregions, 4.1 and 4.2

- Subregion 4.2 governs binding to the -35 box


of the promoter
Structure and Function of σ
Perplexing fact that σ by itself does not bind to promoters
without the core, even if σ regions 2.4 and 4.2 are important
for binding to the -10 and -35 boxes

Region 1.1 prevents σ from binding to DNA in the absence


of core.