Download E. coli

Transcription overview
DNA is the template for transcription
RNA chain growth is 5’ to 3’
Prokaryotic promoters
Prokaryotic promoters
Sigma factor stimulates tight bonding between
RNA polymerase and promoter
The carboxy terminal domain (CTD) of a binds
to the UP element of a promoter.
Overview of transcription initiation
Included: E.coli RNA pol; a
strong promoter; and heparin
Lane 1: no DNA
Lane 2 ATP only
Lanes 3-7: increasing
concentrations of other three
NTPs
s Stimulates Transcription Initiation
Expt 1: Added [g32P]ATP, or
[g32P] GTP to label first
position of RNA (initiation),
OR used [14C] ATP to label all
C’s in the mRNA (elongation)
and asked if s stimulated
incorporation of the labels.
Result: s appeared to
stimulate both initiation and
elongation.
Expt 2: Allowed a certain amount of initiation to
occur, and then added rifampicin to inhibit any
further initiation. By ultracentrifugation, looked
at lengths of mRNAs produced with and without
s.
Result: The presence/absence of s made no
difference to the mRNA lengths, which would
have NOT have been the case if s stimulated
elongation.
Conclusion: s stimulates initiation but not
elongation of transcription.
Re-use of s
- rifampicin, core from
rifampicin-resistant
strain
Experiment done at
low ionic strength.
Label is gamma 32P
labelled NTPs to
measure inititation
only.
Re-use of s
- rifampicin, core from
rifampicin-resistant
strain
Experiment done at
low ionic strength.
Lable is gamma 32P
labelled NTPs to
measure inititation
only.
+rifampicin, core from
rifampicin-resistant strain
Classic View of the s Cycle
Caveat: Evidence for above model might be artifactual; due to harsh separation
conditions of initiation vs. elongation versions of RNA Pol
Recent evidence: s may stay associated with RNA Pol even during elongation.
One model is that the s cycle is really just a shifting of position of s so that it
becomes more loosely associated with RNA Pol.
The Stochastic Model
FRET analysis of sigma movement relative to the DNA
FRET analysis of sigma movement (first three
nucleotides)relative to the DNA
Trailing edge
Leading edge
Local DNA Melting at the Promoter
•RNA Pol-promoter complexes are more stable at elevated
temps. suggesting that local melting occurs upon tight binding
of RNA Pol to DNA
•E. coli RNA Pol binding to T7 gene promoters caused a
hyperchromic shift in the A260 of DNA: indicative of DNA strand
separation
•Gamper and Hearst expt. indicates that RNA Pol melts a short
region of DNA (171 bp) form a transcription bubble that
moves with the polymerase as it transcribes DNA. This exposes
the template as RNA Pol moves along it.
Figure 6.19
How large is the bubble?
(Gamper and Hearst experiment
1.6 superhelical turns per
polymerase = ca. 17 bp
b subunit and phosphodiester bond formation
Rifampicin inhibits transcription initiation. The b subunit of
a rifampicin-resistant strain confers rifampicin resistance when
added to the other subunits that come from a rifampicin
sensitive strain. Therefore, b is important for initiation.
Affinity labeling RNA Pol at its active site
The b subunit was labeled in this experiment indicating that
it is near the active site in RNA Pol where phosphodiester
bonds are formed.
“Walking the polymerase”
b and b’ are involved in DNA-binding
-electrostatic
-involves catalytic site
on or near b and melted
DNA
-weak interaction
-hydrophobic interaction
-involves b and b’
-strong interaction
RNA product of transcription forms RNA-DNA
hybrid with DNA template strand
•RNA-DNA hybrid within elongation complex extends
from the -1 position to -8 or -9 to the 3’ end of the
nascent RNA
•Processivity of transcription depends on at least 9 bp
RNA-DNA hybrid
Structure of Core RNA Polymerase
•Looks like open crab claw: One half of claw made
mainly of b and other half made primarily of b’
•Channel between two parts of the claw is presumably
where DNA passes through
•Three Asp residues at active site coordinate Mg2+ and
are critical for catalysis
Structure of RNA Polymerase Holoenzyme
• Extensive interface between b and the s and b’
subunits.
• may help pry open claw so that it can bind DNA.
• Disordered loop of s appears to approach active site
and lies in exit channel for RNA product.
• Proximity to active site may mean s helps form first
phosphodiester bond; could explain in part its role in
initiation.
• Blocking of exit channel by loop or linker of s may be
why abortive transcripts are made.
Structure of RNA Pol holoenzyme bound to DNA
•DNA mainly bound to s subunit.
•Three highly conserved aromatic amino acids
of s are implicated in promoter melting.
•Template strand of DNA threads through
channel containing active site.
Simple view of structure
Rho independent (intrinsic) termination
Rho-dependent termination