Document 6594457

Gene Expression Prokaryotes II & Eukaryotes Chapters 19, Genes X
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• We can combine all activation
and repressible activities in to
four distinct combinations:
• negative inducible,
• negative repressible,
• positive inducible, and
• positive repressible.
Induction and repression can be under positive or
negative control
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houghton/Regulatory_models.html
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• Binding of repressor at the operator stimulates binding of RNA
polymerase at the promoter but precludes transcription.
• It also opens up the “activator” site for binding of CAP the “Catabolite
Activator Protein” to bind…...and as soon as lactose is present the
system is primed to go!!!
FIGURE 21: Repressor can make a loop in DNA
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• We can combine all activation
and repressible activities in to
four distinct combinations:
• negative inducible,
• negative repressible,
• positive inducible, and
• positive repressible.
Induction and repression can be under positive or
negative control
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Transcriptional Termination Can Be a Regulatory
Event
Rho terminates
transcription
How Does Rho Factor Work?
• Rho factor is a protein that binds to nascent RNA and tracks
along the RNA to interact with RNA polymerase and release it
from the elongation complex.
• rut – An acronym for rho utilization site, the sequence of RNA
that is recognized by the rho termination factor.
• polarity – The effect of a mutation in one gene in influencing the
expression (at transcription or translation) of subsequent genes in
the same transcription unit.
• antitermination complex – Proteins that allow RNA polymerase
to transcribe through certain terminator sites.
Rho can terminate when a
nonsense mutation removes
ribosomes
Antitermination Can Be a Regulatory Event
• An antitermination complex allows RNA polymerase to read
through terminators.
Action at a terminator controls transcription
Antitermination Can Be a Regulatory Event
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Competition for Sigma Factors Can Regulate Initiation
• The activities of the different sigma factors are regulated by different
mechanisms.
• anti-sigma factor – A protein that binds to a sigma factor to inhibit its ability
to utilize specific promoters.
E. coli has several sigma factors
Alternative Regulatory Mechanisms Through
Alternative Sigma Factors…….
The mode of control of sigma54 (the gene product of ntrA or rpoN) is achieved, because (unlike
sigma70) sigma54 cannot function alone -it requires interaction with another protein NtrC (NRI), which
is the gene product of the ntrC gene. Moreover, it is not just the NtrC (NRI) that is required, because
NRI has to be activated into NRI -phosphate by becoming phosphorylated.
NRI is a DNA binding protein which, when phosphorylated binds to specific sequences of DNA and
confers initiation activity on sigma54, promoting the polymerase's ability to form the Rpol/promoter
"open complex". These binding sites do not have to be proximal to the promoter...protein
interactions at a distance!!!
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Alternative Regulatory Mechanisms Through
Alternative Sigma Factors…….
The mode of control of sigma54 (the gene product of ntrA or rpoN) is achieved, because (unlike
sigma70) sigma54 cannot function alone -it requires interaction with another protein NtrC (NRI),
which is the gene product of the ntrC gene. Moreover, it is not just the NtrC (NRI) that is required,
because NRI has to be activated into NRI -phosphate by becoming phosphorylated.
NRI is a DNA binding protein which, when phosphorylated binds to specific sequences of DNA and
confers initiation activity on sigma54, promoting the polymerase's ability to form the Rpol/promoter
"open complex". These binding sites do not have to be proximal to the promoter...protein
interactions at a distance!!!
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The mode of control of sigma54 (the gene product of ntrA or rpoN) is achieved, because (unlike
sigma70) sigma54 cannot function alone -it requires interaction with another protein NtrC (NRI), which
is the gene product of the ntrC gene. Moreover, it is not just the NtrC (NRI) that is required, because
NRI has to be activated into NRI -phosphate by becoming phosphorylated.
NRI is a DNA binding protein which, when phosphorylated binds to specific sequences of DNA and
confers initiation activity on sigma54, promoting the polymerase's ability to form the Rpol/promoter
"open complex". These binding sites do not have to be proximal to the promoter…
…..protein interactions at a distance!!!
The question now is how does NRI become phosphorylated? Through the action of NRII of
course, which is a membrane bound kinase that is able to self phosphorylate and -in response to
LOW levels of NH4+ in the cell can transfer that phosphate to NRI
Herein, finally lies the connection between specific transcriptional initiation factors and levels of
nitrogen in the cell.
NRII is the gene product of ntrB (glnL in E. coli), and relates to ntrC in that it is a member of the
same operon -as is glutamine synthetase (glnA), which is responsible for converting glutamate
into glutamine in the presence of NH4+.
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Eukaryotic Transcription.... Similar Themes, But a
Little Different
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h;p://biology200.gsu.edu/
houghton/Regulatory_models.html
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Eukaryotic Transcription.... Similar Themes, But a
Little Different
• In eukaryotes chromatin must be opened before RNA polymerase
can bind the promoter.
• Multiple DDRPolymerases
• basal transcription factors – Transcription factors required by RNA
polymerase II to form the initiation complex at all RNA polymerase II
promoters
– These Factors are identified as TFIIX, where X is a letter.
Eukaryotic RNA Polymerases Consist of Many
Subunits
• RNA polymerase I synthesizes rRNA in the nucleolus.
• RNA polymerase II synthesizes mRNA in the nucleoplasm.
– heterogeneous nuclear RNA (hnRNA) – RNA that comprises
transcripts of nuclear genes made primarily by RNA polymerase II;
it has a wide size distribution and variable stability.
• RNA polymerase III synthesizes additional “small RNAs” in the
nucleoplasm.
Eukaryotic RNA Polymerases Consist of Many Subunits
RNA polymerase II from yeast
has >10 subunits
•
All eukaryotic RNA polymerases have
~12 subunits and are complexes of
~500 kD.
•
Some subunits are common to all three
RNA polymerases.
•
The largest subunit in RNA Pol II has a
CTD (carboxy-terminal domain)
consisting of multiple repeats of a
heptamer.
RNA Polymerase I
Has a “Bipartite Promoter”
• non-transcribed spacer – The region between transcription units in a
tandem gene cluster.
• The RNA polymerase I promoter consists of a core promoter and an
upstream promoter element (UPE)
• The factor UBF1 wraps DNA around a protein structure to bring the core
and UPE into proximity.
Pol I promoters have two sequence components
• TBP is a component of the
positioning factor that is required
for each of the different types of
RNA polymerase to bind their
respective promoters.
• The factor for RNA polymerase II
is TFIID, which consists of TBP
and ~14 TAFs, with a total mass
~800 kD.
FIGURE 08: Polymerases bind via
commitment factors
• RNA polymerase III has two types of promoter sequences
• Internal promoters have short consensus sequences
located within the transcription unit and cause initiation to
occur at a fixed distance upstream.
• Upstream promoters contain three short consensus
sequences upstream of the startpoint that are bound by
transcription factors.
There are three types of pol III
promoters
Type 2 internal promoters use
TFIIIC
Type 1 pol III promoters use TFIIIA/C
•
assembly factors – Proteins that are
required for formation of a
macromolecular structure but are not
themselves part of that structure.
•
TFIIIA and TFIIIC bind to the
consensus sequences and enable
TFIIIB to bind at the startpoint.
•
TFIIIB has TBP as one subunit and
enables RNA polymerase to bind.
•
pre-initiation complex – The
assembly of transcription factors at
the promoter before RNA polymerase
binds in eukaryotic transcription.
•
RNA polymerase II requires general transcription factors (called TFIIX) to initiate transcription.
•
RNA polymerase II promoters frequently have a short conserved sequence Py2CAPy5 (the
initiator Inr) at the startpoint.
•
The TATA box is a common component of RNA polymerase II promoters and consists of an AT-rich octamer located ~25 bp upstream of the startpoint.
•
The downstream promoter element (DPE) is
a common component of RNA polymerase II
promoters that do not contain a TATA box
(TATA-less promoters).
•
A core promoter for RNA polymerase II
includes the Inr and, commonly, either a TATA
box or a DPE.
– It may also contain other minor elements.
A minimal pol II promoter has only two elements
• TBP is a component of the
positioning factor that is required
for each type of RNA polymerase
to bind its promoter.
• The factor for RNA polymerase II
is TFIID, which consists of TBP
and ~14 TAFs, with a total mass
~800 kD.
FIGURE 08: Polymerases bind via
commitment factors
•
RNA polymerase II requires general transcription factors (called TFIIX) to initiate transcription.
•
RNA polymerase II promoters frequently have a short conserved sequence Py2CAPy5 (the initiator Inr) at
the startpoint.
•
The TATA box is a common component of RNA polymerase II promoters and consists of an A-T-rich octamer
located ~25 bp upstream of the startpoint.
•
The downstream promoter element (DPE) is a
common component of RNA polymerase II promoters
that do not contain a TATA box (TATA-less
promoters).
•
A core promoter for RNA polymerase II includes the
Inr and, commonly, either a TATA box or a DPE.
–
A minimal pol II promoter has only two elements
It may also contain other minor elements.
An initiation complex assembles at promoters for RNA polymerase II
Adapted from M. E. Maxon, J. A. Goodrich, and R. Tijan, Genes Dev. 8 (1994):
515-524.
• TBP binds to the TATA box in the minor groove of DNA.
• TBP forms a saddle around the DNA and bends it by ~80°.
FIGURE 09: TBP binds to the narrow groove of DNA
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• Other transcription factors bind to the complex in a defined
order, extending the length of the protected region on
DNA.
• When RNA polymerase II binds to the complex, it initiates
transcription.
FIGURE 13: TFIIB helps
position RNA polymerase II
• TFIIE and TFIIH are required to melt DNA to allow
polymerase movement.
• Phosphorylation of the CTD is required for promoter
clearance and elongation to begin.
• Further phosphorylation of the CTD is required at some
promoters to end abortive initiation.
Initiation Is Followed by Promoter Clearance
and Elongation
• The histone octamers must be temporarily modified during
the transit of the RNA polymerase.
• The CTD coordinates processing of RNA with transcription
– Phosphorylation of the CTD is required for promoter clearance and
elongation to begin. – Further phosphorylation of the CTD is required at some promoters to
end abortive initiation.
• Transcribed genes are preferentially repaired when DNA
damage occurs.
• TFIIH also provides the link to a complex of repair
enzymes.
•
DNA-directed RNA polymerase II subunit RPB1 - an enzyme that in humans is encoded by the POLR2A
gene. RPB1 is the largest subunit of RNA polymerase II. It contains a carboxy terminal domain (CTD)
composed of up to 52 heptapeptide repeats (YSPTSPS)
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• An enhancer activates the promoter nearest to itself, and
can be any distance either upstream or downstream of the
promoter.
• A UAS (upstream activating sequence) in yeast
behaves like an enhancer, but works only upstream of the
promoter.
FIGURE 15: Enhancer action is
independent of location
FIGURE 09: RNA polymerase exists as a holoenzyme
FIGURE 03: Architectural proteins control the structure of DNA
An activator is a TF that activates transcription. A
classical “true” TF has a DNA binding domain and a
transactivation domain that contacts the basal
transcription complex.
•Activators bound to a
promoter can recruit
basal transcription factors.
•Alternatively, bound
activators may transactivate
basal TFll components already
bound near the start site.
•Different associated factors can be
present in the basal transcription
complex at different promoters.
Bipartite nature of transcription factors (activators)separate DNA binding and activator domains
Basic premise of the
yeast 2-hybrid system
Some activators have only a DNA binding domain.
• RNA polymerase may be
associated with various
alternative sets of
transcription factors in the
form of a holoenzyme
complex.
RNA polymerase exists as a
holoenzyme
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