Download Promoters

Chapter 6
The Transcription
Apparatus of
Prokaryotes
RNA Polymerase Structure
• The subunit content of an RNA polymerase
holoenzyme is ’, , 2, ω and .
• ’ :160 kD; : 150 kD; : 40 kD; 70: 70
kD ; ω: 10 kD
• 3 regions of conservation: -35, -10 and the
length of spacer 17 bp  1 bp
Fig. 6.1
Sigma as a Specificity Factor
• The E. coli enzyme is composed of a core,
which contains the basic transcription
machinery, and a σ factor, which directs
the core to transcribe specific genes.
Promoters
• The polymerase binding sites, including the
transcription initiation sites, are called
promoters.
RNase-resistance
holoenzyme
Core
polymerase
Binding of RNA polymerase to
Promoters
• 3H-labeled T7 DNA to bind to E. coli core
polymerase (blue) or holoenzyme (red).
• Next they added an excess of unlabeled T7
DNA so that any polymerase that
dissociated from the labeled DNA would be
likely to re-bind to unlabeled DNA
• Filter the mixtures through NC at various
times to monitor the dissociation.
T ½= 30- 60 hrs
T ½= less than 1 min
• High temperature promotes DNA melting
(strand separation), this finding is consistent
with the notion that tight binding involves
local melting of the DNA.
More stable
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 sigma-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 complex and is
only in the presence of sigma.
Promoter Structure
• Prokaryotic promoters contain two regions
centered at –10 and –35 base pairs upstream
of the transcription start site. In general, the
more closely regions within a promoter
resemble these consensus sequences, the
stronger that promoter will be.
enhancer
30 X increase of activation
Transcription Initiation
• Carpousis allowed E. coli RNA polymerase to
synthesize 32P-labeled RNA in vitro using a DNA
containing lac UV5 promoter, heparin to bind any
free RNA polymerase
• Heparin: negatively charged polysaccharide that
competes with DNA in binding tightly to free
RNA polymerase
• Abortive transcripts would be up to 9-10 nt in size.
Lane 1: no DNA;
lane 2, ATP only;
lane 3-7: ATP with
concentrations of
CTP, GTP, and
UTP increasing by
two-fold in each
lane.
• Because the heparin in the assay prevented
free polymerase from re-associating with
the DNA, this result implied that the
polymerase was making many small,
abortive transcripts without ever leaving the
promoter.
• The abortive transcripts up to 9 to 10 nt in
size.
Fig. 6.9
The Functions of sigma
•  stimulates initiation, but not elongation,
of transcription.
•  can be re-used by different core
polymerases, and the core, not , governs
rifampicin sensitivity or resistance.
• Rifampicin: blocks prokaryotic transcription
initiation but not elongation.
The incorporation
of the [14C]ATP
measured bulk
RNA synthesis;
the incorporation
of the -32P
nucleotide
measured initiation
Even though sigma
seems to stimulate both
initiation and
elongation, it was due
to an indirect effect of
enhanced initiation
Further experiment
•  stimulates initiation, but not elongation, of
transcription was further demonstrated by the use
of
“Rifampicin”( blocks prokaryotic transcription
initiation but not elongation). They held the
number of RNA chains constant and then use
ultracentrifugation to measure the length of the
RNA in the presence and absence of sigma.
Experiment demonstrate that sigma can be
recycled.
• The key was to run the transcription
reaction at low ionic strength, which
prevent RNA polymerase core from
dissociating from the DNA template at the
end of a gene.
The number of RNA
chain
Constant by allowing a
certain amount of
initiation to occur and
then blocking any
further initiation by
rifampicin; then add
rifampicin-resistant core
polymerase
- rifampicin
+ rifampicin
Reuse of 
• During initiation  can be recycled for additional use
in a process called the  cycle
• Core enzyme can release  which then associates
with another core enzyme
6-25
Sigma may not associate from core During
Elongation
• Fluorescence resonance energy transfer
(FRET): two fluorescent molecules close to
each other will engage in transfer of resonance
energy, and the efficiency of this energy
transfer will decrease rapidly as the two
molecules move apart.
Summary
• The sigma factor changes its relationship to the
core polymerase during elongation, but it may
not dissociate from the core. Instead it may
just shift position and become more loosely
bound to the core.
Local DNA melting at the promoter
• When A is base-paired with T, the N1
nitrogen of A is hidden in the middle of the
double helix and is protected from
methylation
• S1 nuclease can cut the DNA at each of the
unformed base pairs because these are local
single-stranded regions.
Lane R+S+ shows the
results when both RNA
polymerase ( R) and S1
nuclease (S) were used.
On binding to a
promoter, RNA
polymerase causes the
melting of at least 10 bp,
Structure of Sigma
• Sigma 70 family: There are four conserved
regions in sigma 70 family proteins.
• The best evidence for the functions of these
regions shows that sub-regions 2.4 and 4.2
are involved in promoter –10 box and –35
box recognition.
• Region 1: found only in the primary sigmas
( sigma 70 and 43)
• Region 2: most highly conserved sigma
region, 2.4: -10 box binding
• Region 3: helix-turn-helix DNA binding
domain
• Region 4: 4.2 : -35 box binding
Fig. 6.20
Fig. 6.21
P: lacking the tac promoter
 In this experiment contains only the region 4, not region 2.
Because pTac DNA competes much better than P DNA, they
concluded that the fusion protein with region 4 can bind to the tac
promoter.
The role of the -subunit in UP element
recognition
• The RNA polymerase -subunit has an
independently folded C-terminal domain that
can recognize and bind to a promoter’s UP
element. This allows very tight binding
between polymerase and promoter.
•α subunit response to activator, repressor,
elongation factor and transcription factors
-235 polymerase: missing 94 C-terminal amino acid of the
 subunit
-88: wild type promoter; SUB: irrelevant sequence instead; -41:
deletion UP
In vitro transcription. What is the conclusion you get from this
experiment?
The bold brackets
indicate the footprints in
the UP element caused
by the -subunit, and
the thin bracket
indicates the footprint
caused by the
holoenzyme.
Elongation
Core Polymerase Functions in Elongation
• The role of β in phosphodiester bond formation :
The core subunitβ binds nucleotides at the active
site of the RNA polymerase where phosphodiester
bonds are formed. Rifampicin can block initiation
by preventing the formation of that first bond.
• The core subunit β’can bind weakly to DNA by
itself in vitro. In fact, both β andβ’bind to DNA as
indicated by different experiments.
The affinity-labeling
reactions: First, add reagent I
to RNA polymerase. The
reagent binds covalently to
amino groups at the active
site. Next, add radioactive
UTP, which forms a
phosphodiester bond (blue)
with the enzyme-bound
reagent I. This reaction
should occur only at the
active site, so only that site
becomes radioactively
labeled.
Labeled the active site as mentioned above, then separate the
polymerase subunits to identify the subunits that compose the active
site
Electrostatic interaction , 
Hydrophobic interaction
 and ’
Termination of Transcription
• Rho-independent Termination: inverted
repeats and Hairpins, a string of Ts in the
nontemplate strand
rU-dA have a
melting temperature
20 degree lower than
rU-rA or rA-dT pairs
An assay for attenuation
• If attenuation works, and transcription
terminates at the attenuator, a short 140-nt
transcript should be the result.
• When change the string of eight T’s in the
nontemplate strand, creating a trp a1419
mutant, attenuation was weakened.
• This result is consistent with the weak rUdA pairs are important in termination.
TTTTGAA: trp a1419, attenuation weakened
IMP: inosine monophosphate, a GMP analogue, weaken basepairing in the hairpin, I=C weaker than GC pair
The essence of a bacterial terminator
is twofold
• 1. Base-pairing of something to the transcript to
destabilize the RNA-DNA hybrid
• 2. Something that causes transcription to pause
• A normal intrinsic terminator satisfies the first
condition by causing a hairpin to form in the
transcript, and the second by causing a string of
U’s to be incorporated just downstream of the
hairpin.
• Rho-dependent Termination: consist of an
•
•
•
•
inverted repeat, which can cause a hairpin to form in
the transcript, but no string of Ts.
Rho affects chain elongation, but not initiation.
Rho causes production of shorter transcripts.
Rho is an RNA helicase, composed of 6 identical
subunits, each subunit has an RNA binding domain
and ATPase domain
Rho releases the RNA product from the DNA
template.
Chapter 7
Operons: Fine Control of
Prokaryotic
Transcription
The lac Operon
• Lactose metabolism in E.coli is carried out
by two enzymes, with possible involement
by a third. The genes for all three enzymes
are clustered together and transcribed
together from one promoter, yielding a
polycistronic message.
• The lac Operon: It contains three
structural genes – genes that code for
proteins : -galactosidase (lacZ),
galactoside permease (lacY), and
galactoside transacetylase (lacA).
• They all are transcribed together on one
messager RNA, called a polycistronic
message, starting from a single promoter.
• Negative Control of the lac Operon
• Repressor-operator Interactions
• Lac repressor binds to lac operator was
demonstrated by filter-binding assay.
The repressor is an allosteric protein:
one in which the binding of one
molecular to the protein changes the
shape of a remote site on the protein
and alter its interaction with a second
molecule. Inducer: 1st molecule;
operator: 2nd molecule
Constitutive mutants had a defect in the gene (lacI)
Constitutive mutant
Because it is
dominant
only with
respect to
genes on the
same DNA
Constitutive and dominant
Because the
mutant repressor
will bind to
operators even in
the presence of
inducer or of WT
repressor
The mechanism of Repression
• RNA polymerase can bind to the lac
promoter in the presence of the repressor.
The function of the repressor appears to
inhibit the transition from the nonproductive synthesis of the abortive
transcripts to real, processive transcription.
Assaying the binding between lac operator and lac repressor
• Cohen and colleagues labeled lacO-containing
DNA with 32P and added increasing amounts of
lac repressor
• They assayed binding between repressor and
operators by measuring the radioactivity attached
to NC.
• Only labeled DNA bound to repressor would
attach to NC.
• IPTG: prevents repressor-operator binding.
Mutant O with low affinity
Wild type operator
Nonsense DNA
1. Incubation of a DNA
fragment containing
the lac promoter with
(lanes 2 and 3) or
without (lane 1) lac
repressor (LacR).
2. After repressoroperator binding had
occurred, they added
RNA polymerase.
After 20 minutes for
OC to form, they
added heparin and all
components except
CTP.
3. Finally, after 5 more
minutes, they -32P
CTP alone or with the
inducer IPTG then wait
for 10 minutes for
RNA synthesis. The
result showed that
transcription
occurred even when
repressor bound to
the DNA before
polymerase could,
repressor did not
prevent polymerase
from binding and
forming an open
promoter complexes.
( but the condition is
nonphysiological
conditions, too much
proteins)
Effect of lac repressor on dissociation of
RNA polymerase from the lac promoter
• Record made complexes between RNA
polymerase and DNA containing the lac promoteroperator region
• They allowed the complexes to synthesize
abortive transcripts in the presence of a UTP
analog fluorescently labeled.
• As the polymerase incorporates UMP from this
analog into transcripts, the labeled pyrophosphate
released increases in fluorescence intensity.
( condition likely in vivo)
• The latest evidence supports the repressor, by
binding to the operator, blocks access by the
polymerase to the adjacent promoter.
Effects of mutations in the three lac
operators
• WT or mutant lac operon on  phage
• Infect and lysogenize E. coli
• Assay for -galactosidase in the presence or
absence of IPTG
+IPTG/-IPTG
Positive Control of the lac Operon
• It is mediated by a factor called catabolite
activator protein (CAP) in conjunction with
cyclic AMP, to stimulate transcription.
• Sensed the lack of glucose, increase of
cAMP.
• CAP is dimeric and binds to 22 bp operator
sequences, accelerates the initiation of
transcription at these promoters.
Once the first phosphodiester bond forms, the polymerase is
resistant to rifampicin inhibition until it re-initiates.
CAP binding sites in the lac, gal and ara operons all contain the
sequence TGTGA
Lac operon has remarkably weak promoter , -35 box
Mechanism of CAP Action
• The CAP-cAMP complex stimulates transcription
of the lac operon by binding to an activator site
adjacent to the promoter and helping RNA
polymerase to bind to the promoter. This closed
complex then converts to an open promoter
complex. CAP-cAMP causes recruitment through
protein-protein interactions, by bending the DNA,
or by a combination of these phenomena.
Binding of CAP-cAMP to the activator site
does cause the DNA to bend
• When a piece of DNA is bent, it migrates more
slowly during electrophoresis.
• The closer the bend is to the middle of the DNA,
the more slowly the DNA electrophoreses.
• Actual electrophoresis results with CAP-cAMP
and DNA fragments containing the lac promoter
at various points in the fragment, dependent on
which restriction enzyme was used to cut the DNA.
Fig. 7.19
Fig. 7.20
Tryptophan’s Role in Negative
Control of the trp Operon
• The trp Operon contains the genes for the
enzymes that E. coli needs to make the amino acid
tryptophan.
• The trp operon responds to a repressor that
includes a corepressor, tryptophan, which signals
the cell that it has made enough of this amino acid.
The corepressor binds to the aporepressor,
changing its conformation so it can bind to the trp
operator, thereby repressing the operon.
Fig. 7.28
5 structural genes
High conc. of tryptophan is a signal
to turn off the operon
Trp repressor
Control of the trp Operon by
Attenuation
• Because of the weak repression of the trp operon,
another extra control called attenuation exists.
• Attenuation imposes an extra level of control on
an operon, over and above the repressor-operator
system. It operates by causing premature
termination of transcription of the operon when
the operon’s products are abundant.
Figure 7.30 Two Structures
available to the leaderattenuator transcript.
Riboswitches
• Is a region in the 5’-UTR of an mRNA that contains
two modules: an aptamer that can bind a ligand, and
an expression plateform whose change in
conformation can cause a change in expression of the
gene.
• FMN can bind to an aptamer in a riboswitch called
the RFN element in the 5’-UTR of the ribD mRNA.
• Upon binding FMN, the base pairing in the
riboswitch changes to create a terminator that
attenuates transcription.
Fig. 7.34
Fig. 7.35