Download Transcription in prokaryotes Elongation and termination

Transcription in prokaryotes
Elongation and termination
After initiation the σ factor leaves the scene.
Core polymerase is conducting the elongation of the chain.
The core polymerase contains main nucleotide synthesizing
machinery:
β - involved in phosphodiester bond formation
β and β’ – involved in DNA binding
α- has several actions, including assembly of polymerase.
Role of subunits were first investigated in 1970 by Walter Zillig
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Role of core subunits
Approach: separating core into polypeptides and combining
them back to reconstitute the functions.
Separation: electrophoresis on cellulose acetate with the
presence of urea.
Urea is denaturing agent that can separate individual
polypeptides from the complex.
Advantage: It is a mild agent, easy to remove.
Separation and reconstitution allowed to figure out which
subunit determined resistance or sensitivity to rifampicin.
α+β+’σ from rif sensitive bacterium + β from rif resistant
= rif resistant polymerase
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β subunit is involved in PDB formation
Approach: used affinity labeling experiment
Idea:
•Label an enzyme with a derivative of a normal substrate that
can be cross-linked to protein.
•The affinity reagent can seek out and label the active site of
the enzyme.
•The enzyme can be dissociated, and one can see the
subunit that the tag was attached to.
•14 different reagents were used – all ATP and GTP analogs.
Outcome: β subunit is the only core subunit labeled by the
affinity reagents, suggesting that it is responsible for
nucleotide binding and PDB formation.
Conclusion: β subunit is involved in PDB formation
One of different reagents that were
used. The ATP analog.
Steps:
1. Add analog to RNA polymerase.
It covalently binds to amino
groups at active site
2. Add radioactive UTP, it forms PDB
with the enzyme-bound reagent.
3. Reaction occurs at the active site
which becomes labeled.
Outcome: β subunit is the only core
subunit labeled by the affinity
reagents, suggesting that it is
responsible for nucleotide binding
and PDB formation.
Fig. 6.32
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Conclusion:
Subunit β binds nucleotides at the active site of the RNA
polymerase where PDBs are formed
The σ factor is also near the nucleotide-binding site, at
least during initiation.
β and β’ and DNA binding
Studies of E. Nudler, 1996
Cross-linking labeled DNA to polymerase
They have concluded:
 β and β’ are involved in DNA binding
Two binding sites are present
An upstream, weak site where melting occurs, with
electrostatic forces predominating
Downstream strong site where hydrophobic forces bind
DNA and protein together
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Role of α subunit in polymerase assembly
•The α Subunit recognizes UP elements
•It also interacts with activator proteins that bind outside of
promoter
•The N terminal domain plays an important role in
transcription by helping to organize the polymerase.
How?
Order of assembly:
α→α2→ α2 β →α2ββ’ - possibly α plays central role?
Role confirmed by studies involving mutant E. coli strains
Role of α subunit was confirmed by studies involving
mutant E. coli strains
•Mutants with deletions at C-terminus of α were studied
•Mutant proteins were terminated at aa 150,176, 235, 256 and
296.
•Wt protein terminates at 329.
Outcome: up to 94aa could be removed from C-terminus w/o
loss of function
Conclusion:
Assembly function is in the N-terminus.
The points of contact between the α -NTD and the β and β’
subunits lie on the opposite side of the α -NTD dimmer from
the dimmer’s C-terminal domains.
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Elongation
The elongation complex
Experiments with RNA-DNA hybrids
•The RNA-DNA hybrids within the elongation complexes
extend from position –1 to –9,-8 with respect to the 3’end of
the emerging RNA.
•The processivity of transcription depends upon the DNARNA hybrid at least 9bp long.
Fig. 6.49
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From Genes VII web resource
www.oup.com/genesvii
Termination
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RNA pol moves along the DNA synthesizing RNA until it
meets a terminator (t) sequence.
At this point the polymerase:
•Stops adding nucleotides
•Releases the completed product
•Dissociates from the template.
NB – we do not know in which order the last two happen.
Termination requires that all hydrogen bonds holding
DNA-RNA hybrid together must be broken, after which the
DNA duplex reforms.
Termination point –
Is difficult to define the term. point at the RNA that has been
synthesized in the living cell.
Because it is always possible that the 3’ end of the molecule
has been generated by cleavage of the primary transcript, and
therefore does not represent the actual site at which pol was
terminated.
The best identification of the terminator sites is provided by in
vitro systems.
But: because ability of the enzyme to terminate is strongly
influenced by parameters such as ionic strength, the term point
in vitro does prove that the same point is a natural terminator.
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Anti-termination – will cause enzyme to continue past
the t sequence – the read-through.
The hairpin structure
From GenesVII web resource
www.oup.com/genesvii
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Termination is viewed not as a simple event to stop
transcription, but as an opportunity to control gene expression.
Termination, as initiation, is a subject to specific control.
Parallels between initiation and termination:
•Both require breaking of hydrogen bonds
•Both requires additional proteins to interact with core
enzyme.
Differences:
•Initiation relies solely on interaction between DNA and pol
•Termination involves recognition of signals in the transcript
by RNA pol and ancillary enzymes
Sequences of prokaryotic terminators show no similarities
beyond the point at which the last base is added to the RNA.
The responsibility for termination is with the sequences
already transcribed by RNA polymerase.
It relies on scrutiny of the template or product that
polymerase is currently producing.
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Terminators:
Core enzyme can terminate in vitro at certain sites in
absence of any other factors. These sites are called
intrinsic terminators.
Rho-dependent terminators are defined by the need of
addition factor (ρ) in vitro.
Intrinsic terminators include
•palindromic regions
•The stem-loop structure with a G-C rich region.
•a run of U-nucleotides.
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What is the effect of hairpin?
•Probably the hairpins cause the polymerase to slow or
perhaps to pause the synthesis.
•Pausing creates the opportunity for termination.
The length of the pause varies, but at a typical terminator it
lasts ~60 seconds.
Role of the U-run
•A U-string is necessary for polymerase to dissociate from
the template when it pauses at the hairpin.
•The rU-dA RNA-DNA hybrid is unusually weak bp structure.
•Termination usually takes place at any of several positions
toward or at the end of U-run, as though the enzyme
‘stutters’ during termination.
Importance of the U-run was confirmed by making
deletions that shorten the stretch – although the polymerase
paused, it could not terminate.
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The model
Fig. 6.51
The Rho factor – how does it work
The Rho factor:
•Functions in E. coli solely at the termination stage.
•46kD protein, acts as hexamer.
•Function: ancillary factor of RNA polymerase.
•Max activity displayed in vitro when present at conc. ~10%
of RNA polymerase.
•E. coli has relatively few Rho factors.
•The sequences required for Rho are upstream of
terminator, are 50-90 bp long.
•Common feature – the RNA is rich in C and poor in G
residues.
•EG: C-41%, g-14%.
Rule: efficiency of Rho terminator depends upon
increases in length of the C-rich/G-poor region.
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The Rho factor – how does it work
From GenesVII web resource
www.oup.com/genesvii
The Rho factor – how does it work
Rho factor:
•Has ATPase activity
•Binds to RNA
•An individual Rho factor acts processively on the RNA
strand
•The ‘hot pursuit’ model
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The Rho factor –
how does it work
Fig. 6.56
The Rho
factor – how
does it work
From
GenesVII
web resource
www.oup.com
/genesvii
15
The action of Rho may create a link between transcription
and translation
Rho first must have access to a binding sequence of RNA
Must be able to move along the RNA
Either of both of these conditions may be prevented if
ribosomes are translation an RNA.
Thus – the ability of Rho factor to reach a terminator depends
upon what is happening in translation
The model to explain this puzzling phenomenon:
•In some cases, nonsense mutation in one gene of
transcription unit prevents the expression of subsequent genes
in the unit.
•This effect is called polarity.
•A common cause is the absence of mRNA corresponding
to the distant parts of the unit.
•Example:
Suppose there are Rho-dependent terminators within the
transcription unit, that is before the terminator that is usually
used.
•Normally these early terminators are not used, because the
ribosomes prevent Rho from reaching the RNA polymerase.
•But nonsense mutation releases the ribosomes, so that rho
is free to attach and move along the RNA, enabling is to
react with RNA polymerase.
•Result: enzyme is released, distal parts are not transcribed.
???Why there should be internal terminators???
Perhaps they are simply sequences that by coincidence
mimic the usual rho-dependent terminator.
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From
GenesVII
web resource
www.oup.com
/genesvii
Phenomenon of antitermination:
•Antitermination proteins allow RNA polymerase to
transcribe through certain termination sites.
•Is used as a control mechanism in phage regulatory
circuits and bacterial operons.
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References:
R. Weaver. Molecular Biology
B. Lewin. Genes VII, 2000, web resource:
www.oup.com/genesvii
Reading:
Elongation.
Pages 154-171.
W/o extensive details of crystallography studies.
Termination:
Pages 171-178
W/o details of figure 6.52
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