RNAP
-catalytically active CORE with subunit composition ββ’α2
-core can synthesize RNA, but can not initiate transcription because does not recognize
promoters
-promoter selectivity is conferred by a sigma factor, σ
- ββ’α2 σ constitutes RNAP HOLOENZYME
-most promoters use a major sigma factor, known as σ70 in E. coli
-ALTERNATIVE SIGMA FACTORS are used to control specialized subsets of genes
-all subunits of E. coli RNAP have been overproduced and can be purified for in vitro
reconstitution of core and holoenzymes containing defined genetic alterations; has facilitated
investigation of the in vitro transcription properties of RNAP containing alterations that would
be lethal in vivo
GENE
SUBUNIT
rpoA
alpha (α)
rpoB
beta (β)
rpoC
rpoD
beta (β’)
sigma (σ)
RNAP
FUNCTION
STOICHIOMETRY
2
-initiates RNAP assembly
-interacts with DNA, transcription factors, and RNA during
initiation and elongation
-2 protein domains (CTD & NTD) linked by flexible linker
-NTD contains sites for dimerization & assembly
-CTD contains sites for DNA, RNA, & transcription factor
interaction
-can bind to DNA upstream of –35 σ contacts (-40 to –65)
--40 to –65 is called an UP element because α binding increases
RNAP affinity for promoter
-UP elements important for expression of highly expressed
genes
-flexible linker allows for diverse interactions between α and
upstream bound activator proteins
1
-catalytic center of enzyme
-binds initiating nucleoside triphosphates and growing RNA
chain
-clamps RNAP to DNA template during elongation
-mediates recognition of factor-independent termination signals
1
-same as for β, above
1
-specifies promoter to which RNAP binds
TRANSCRIPTION INITIATION REVIEW
-several biochemical steps
Step 1
RNAP binding to promoter
Step 2
Melting of DNA helix/isomerization
of RPc
Conversion of RPo to initiating
complex by synthesis of first
phosphodiester bond
Abortive transcription/idling
Promoter clearance and conversion
to processive, elongating complex
Step 3
Step 4
Step 5
Closed complex
(RPc)
Open complex
(RPo)
Initiating complex
(RPi)
RPi
Elongating
complex (RPe)
PROMOTER STRUCTURE REVIEW
-distinct promoter elements involved in different steps of
transcription initiation
Promoter element
-35
-10
Spacer
USR/Up element
DSR
Function
S binding
S binding; isomerization
Binding; isomerization
A binding & RPc formation;
promoter idling/clearance
Promoter idling/clearance
-promoter strength dictated by:
i)
ii)
DNA sequence/presence of consensus elements
Regulatory proteins
-3 different strengths of promoters:
Constitutive
-consensus or near
consensus promoter
-constant rate of
transcription
-may be unregulated
Defective
-one or more
consensus elements
lacking
-low rate of
transcription
-often require
activators
Strong
-strong promoter
sequence &/or
activators
-high rate of
transcription
-often subject to
repression
-10 TATATT
-35 TTGACA
MECHANISMS OF REPRESSION
STERIC HINDRANCE
-“classic” mode of repression
-repressor binding site overlaps promoter elements
-repressor binding prevents RNAP binding/formation of RPc
-eg. LacI O1
-O1 = LacI binding site initially discovered by Jacob & Monod
-now know there are 2 other operators O2, O3
-in absence of O2, O3, O1 still allows for repression of the lac
operon by LacI
-mechanism is by inhibiting RNAP binding
EFFECTS ON DNA
-structure of the DNA is important for transcription initation
-RNAP can contact –35, -10, USR, and DSR simultaneously
-structural studies suggest that this is accomplished by wrapping
the DNA around the molecule
-repressors may change DNA structure in ways that either preclude
RNAP binding completely or prevent all of necessary interactions
with DNA (wrapping)
DNA looping
eg. LacI
-DNA binding studies identified a second LacI operator (O2)
within the lacZ gene (+401)
-sequence gazing identified a third operator, O3, upstream of the
promoter (-92)
-O2 and O3 have low affinity for Lac repressor relative to O1
-are these operators involved in repression?
-test effects of deleting O1, O2, O3 on ability of LacI to repress
Operators
O1
O2
O3
O1+O2
O1+O3
O1+O2+O3
Fold Repression
18
None
None
700
440
1300
-even though affinity for repressor is weak, O2 and O3 contribute
2-3 fold greater repression when present with O1
-how?
-since presence of either O2 or O3 can partly compensate for each
other, propose they are able to cooperate with O1 to repress lac
operon loop formation?
-since each operator binds a repressor dimer, loop formation would
require a tetramer
-mutants of LacI which cannot form tetramers exhibit 60X lower
repression and repression is unaffected by removing O2 or O3
Dimeric repressor
Wild-type
O3+O1
110
90
Tetrameric
Repressor
6700
3900
O2+O1
O1
80
60
1400
140
-suggests that repression occurs through loop formation between
either O1 and O2 or O1 and O3
-mechanism of repression by DNA looping uncertain
-two possibilities:
i)
ii)
formation of DNA loop prevents RNAP binding
formation of DNA loop allows RNAP binding, but
sterically inhibits DNA melting
eg. GalR
-represses gal regulon – enzymes for galactose transport and
metabolism
-represses two promoters P1 and P2 by binding to operators OE (60.5) and OI (+53.5)
-how does repression work?
-Adhya lab-replace one GalR operator with a LacI operator – if
only need occupation of sites for repression then should not make a
difference LOSE REPRESSION
-therefore not simple occupation of sites that causes repression
-if replace both operators with lac operators REPRESSION
-argues that repression requires interaction between operatorbound repressor molecules and DNA loop formation
-additional evidence that this is so insertion of a full turn (or
multiples) between Gal operators does NOT prevent repression,
insertion of 1.5 turns such that Gal repressors would be on opposite
sides of DNA DOES
-mechanism of repression by DNA looping uncertain
-two possibilities:
iii)
iv)
formation of DNA loop prevents RNAP binding
formation of DNA loop allows RNAP binding, but
sterically inhibits DNA melting
Other DNA Structural Changes Causing Repression
-structural changes to DNA in area of promoter can physically
block RNAP binding or prevent subsequent steps in transcription
initiation
-eg. DNA needs to wrap around RNAP
eg. H-NS
-small, abundant protein
-avidly binds bent DNA
-generally functions as a repressor by inhibiting RNAP binding or
strengthening repressor:DNA complexesH-NS
-structural protein involved in packaging bacterial DNA into
chromatin
-down-regulates expression of several genes
-binds to bent DNA to form multimeric nucleoprotein complexes
in which DNA wraps around H-NS
PROTEIN:PROTEIN CONTACTS
-repression of transcription at a stage beyond formation of RPc
-simultaneous binding of repressor and RNAP
-repressor:RNAP interactions lead to inhibition of any step after
RPc to inhibit transcription initiation (Rpo or Rpi formation,
inhibition of promoter clearance)
Inhibiting RPcRpo
eg. MerR
-controls expression of bacterial mercury-resistance operon
(merTPCAD)
-MerR = negative and positive regulator of merTPCAD operon
-MerR binding site in spacer between –35 and –10 region
-Summers 1990 – DNA footprinting shows that MerR binds to
same site in both presence and absence of mercury
-this binding site overlaps with that of RNAP
-MerR:RNAP:DNA complex is heparin sensitive, indicating that
MerR prevents open complex formation
-in presence of mercury, changes in accessibility of –10 region
seen MerR bound to Hg induces a conformational change in the
DNA
-model: MerR bound to Hg changes promoter conformation to
bring together –10 & -35, promoting Rpo formation
eg. GalR
-if looping is prevented by mutation of OI operator or use of
repressors that do not interact with each other, GalR still represses
transcription at P1
Rpo through contacts with the α
-inhibits transition from RPc
subunit of RNAP
-GalR and RNAP bind DNA simultaneously at OE and P1
-this effect requires the C-terminal domain of the α subunit
-model: contact between GalR and RNAP a subunit prevents a
stage in transcription initiation after formation of RPc
inhibiting Inhibiting RPiRPe
eg. lacI
-1987 Straney & Crothers
-RNAP and Lac repressor bind DNA at the same time
-use lacUV5 promoter – very strong mutated promoter that does
not require CAP for transcription (no O2 or O3!)
-when mixed lac repressor + RNAP + lacUV5 promoter complexes containing RNAP + lac repressor
-performed transcription assays on complexes (add labeled rNTPs)
-during abortive initiation typically see an 8mer
-if add a chain terminating rNTP (3’ OMeCTP) , can visualize the
elongation complex, which escapes the promoter and can
synthesize an 11mer that ends in C
-in presence of repressor, abortive transcription is seen (8mers)
-if add IPTG, allow progression to elongation phase (11mer)
-therefore, LacI binds repressor at same time as RNAP, but
prevents it from progressing from Rpi to RPe
-NB: under conditions thought to predominate in the cell, lac
repressor and RNAP are not found on DNA at same time!
ANTI-ACTIVATION
-repression by interference with DNA binding or activity of an
activator protein
-common in eukaryotes, less common in prokaryotes
eg. CytR
-negative regulator of operons that encode proteins that allow
utilization of nucleosides and deoxynucleosides as sole energy
source
-operons also controlled by CAP /CRP (activated when camp is
high/glucose is low)
-CytR works by preventing activation by cAMP-CAP
-at CytR-regulated promoters, there are usually 2 CAP binding
sites
-very weak CytR binding site found between the CAP sites
-how does repression work?
-repression requires cAMP-CAP binding at both sites
-cAMP-CAP and CytR bind cooperatively to CytR-repressed
promoters to form nucleoprotein complex
-repression requires specific CytR:CAP interactions, since aa
substitutions in CAP eliminate repression, but do not affect CAP
activation
-repression does NOT require the DNA binding domain of CytR
-CytR:CAP:DNA nucleoprotein complex prevents access of RNAP
-inducers cytidine and adenosine relieve repression by interrupting
CytR:cAMP-CAP interactions