Elongation/Termination

1
Elongation/Termination First, an overview of
what hold RNAP on DNA during elongation Then
some examples of control.
Note after sigma leaves nusA and sometimes other
factors can join. So far, it is not clear exactly
how they are involved in termination control.
2
This is a model of the transcription complex in
the process of elongation. (The nucleic acid data
comes partlyfrom footprinting and cross-linking)
Transcription in this direction
Rudder helping to keep template DNA melted.
9 bp DNA-RNA hybrid
RNA exit channel, covered by flap
Blue residues - basic Red residues - acidic
A melted base pair is matched to the NTP. The NTP
is added to the 3 end of the RNA. The polymerase
ratchets forward One bp closes behind and a new
one opens ahead.
3
Elongation is processive, but polymerase
sometimes pauses.
When polymerase pauses it has the potential
to terminate transcription This only occurs
when the RNA is released
terminator
Pause sites
promoter
Time after starting a round of transcription
4
These may be paused because the 3 end of the
RNA has slipped out of the enzyme active site.
Footprints at pause sites
These footprints show that the elongation
complex does not always move smoothly down the
DNA during elongation. As RNA synthesis occurs
in these extreme examples the polymerase front
end appears not to have moved.
Footprinting results on stalled complexes from
studies of Krummel and Chamberlin 71 and 72.
The DNase I and KMnO4 footprints of complexes
stalled at positions 20 (top), 23 (middle) and 27
(bottom). The sequence of the DNA template is
shown with the sequence of the transcript below
it. The nucleotides on the template and
nontemplate strands that were protected from
DNase I digestion are shown as bars above and
below the DNA sequence. The region that was
susceptible to KMnO4, and therefore is single
stranded, is shown in the gray box. An underlined
"T" indicates that thymine residue was
susceptible to KMnO4. The data are taken from the
PhD thesis of Krummel 97.
5
Pausing is probably due to misalignment between
the RNA 3' end and the active site. How DNA or
RNA or hybrid sequences cause this is not known.
bind NTP
chemistry
ratchet
RNA
Smooth elongation
RNA end
Misalignment during elongation - backtracking
observed frequently
Active site
(BBA) - Gene Structure and Expression Volume
1577, Issue 2 , 13 September 2002, Pages 224-239
6
Backtracked RNAPs can be re-activated by Gre
factors
Recall that Gre factors enter secondary channel
and position acidic residues at active site of
RNAP. This stabilizes a second Mg setting up
metal-catalyzed cleavage of the RNA (see next
slide).
cleavage re-aligns active center with the 3
end of the RNA. Elongation can resume.
OpalakaDarst - Cell. 2003 Aug 8114(3)335-4
7
Evidence for Gre-induced cleavage via acidic
residues at active site of RNAP
RNA from stalled RNAP
released cleavage products
mutants inhibit cleavage
Other experiments show that GreB increases
elongation rate and suppresses pausing
8
RNAP also transiently stalls and backtracks at
roadblocks (may be common on real chromosomes)
Experiment Block with stuck R1 at limiting RNAP.
Wash away excess free RNAP. Observe backtracking
by gre-induced shortened RNA (80-long). Remove
roadblock with salt and RNAP can re-position
(resistant 84-long)
EMBO J. 2003 Sep 1522(18)4719-2
Note claims that re-activation is assisted by
trailing RNAPs - check if interested
9
Termination will occur when RNA is released
during a pause.
When polymerase pauses it has the potential
to terminate transcription This only occurs
when the RNA is released
terminator
Pause sites
Question What holds the RNA in the elongation
complex?
promoter
Time after starting a round of transcription
10
3 interactions with polymerase keep the RNA in
the elongation complex. In addition the RNA is
bound to the DNA in the hybrid.
Hybrid binding site
2 RNA binding sites
Exit channel
11
What are the signals associated with
terminators? Generally there are 2 types

• A GC-rich partial dyad followed by a stretch that
  codes for oligo uridine.
• The U-stretch is at termination site. The dyad is
  just upstream.
• These signals work primarily through RNA rather
  than DNA.

TTTTTT AAAAAA
DNA
RNA
UUUUUU
Terminator hairpin
U stretch
(there also may be an influence of DNA just
downstream from the U-stretch.)
How do these work to de-stabilize the RNA?
12
Here's the TEC at a potential terminator
The rUdA hybrid is the weakest of all possible
hybrids.
Hybrid binding site is rUdA
RNA binding sites
13
Another view of termination
This state binds the RNA weakly and releases it
--- termination
Figure 1 The mechanism of intrinsic termination.
Termination begins with a brief pause (1.5-2 s)
at the termination point (e.g. the U7 position
of the tR2 terminator) induced by the T-stretch
of the terminator. Three RNAP nucleic acid
binding sites are indicated in the paused
complex HBS (RNADNA hybrid binding site), RBS
('tight' RNA-binding site), and UBS ('weak'
upstream RNA-binding site) Transition from a
paused to a termination complex occurs due to
hairpin formation and involves breaking
protein-RNA contacts in the UBS and the RBS,
partial breaking of contacts in the HBS due to
unwinding of 5-6 bp of the hybrid, irreversible
complex inactivation, and rewinding of the
transcription bubble from behind. NusA
stimulates hairpin formation principally by
weakening the contacts between the potential
ascending loop of the hairpin (the 'upstream
shoulder' of the unfolded hairpin) and the UBS,
and to a lesser extent by prolonging pausing at
the termination point
14
Another signal for termination is the absence of
RNA secondary structure. In this case the RNA
helicase rho binds the RNA and releases it.
Rho is a hexamer ring that can load ssRNA, if it
is available. Most terminators are downstream
from the translation stop codon. This is to
allow increased access of rho to the RNA at this
time.
at the end of a gene
When rho approaches polymerase, it this thought
that the ring closes and then the ATPase pulls
out the RNA.
Anti-termination factors can block rho binding to
RNA
15
Control of termination is very simple Factors
control whether the terminator hairpin forms.
16
Examples of regulators
Trp operon (makes trp) When trp is present the
hairpin forms (do not want more trp made). When
trp absent ribosomes stall and melt the
terminator (get transcription now)
Regulator proteins bind directly and control
hairpin formation. (example coming).
FMN, lysine etc. bind RNA directly and induce
formation of the terminator hairpin. This
prevents unnecessary expression of synthetic
enzymes. First overview of FMN and then lysine
experiments.
17
FMN example
Terminator hairpin
promoter
This operon produces proteins needed for
synthesis of FMN
If enough FMN
If not enough FMN
FMN causes the terminator hairpin to form as
shown in the next slide. Remarkably, FMN binds
RNA and stabilizes a structure that exposes the
hairpin
18
Termination by FMN
FMN - terminator hairpin forms
No FMN - structure forms without terminator
hairpin.
Therefore, terminate transcription only when FMN
is present so dont make proteins to synthesize
more.
19
Examples of termination structures stabilized by
metabolites. All allow formation of the
terminator hairpin. (normally the hairpin
elements are tied up in more stable structures).
20
L-box sequences prevent biosynthesis of lysine
when it is already present.
PNAS October 14, 2003 vol. 100 no. 21
12057-12062 - B. subtilis system
Terminator hairpin
promoter
This operon produces proteins needed for
synthesis of lys
If enough lys
If not enough lys
21
ReadThrough
Termination
Changes in RNA structure can alter termination.
22
Lysine binds RNA directly and leads to less
digestion (red), defining its binding site in the
predicted structure
GENES DEVELOPMENT 172688-2697, 2003
23
The lys-bound structure allows a terminator
hairpin to form
M1 de-stabilizes terminator hairpin and allows
more readthrough
M2 decreases stability of anti-loop. Get more
termination without lysine.
M3 restores stability lost in M2. A bit closer to
normal regulation.
24
Riboswitches are very common in gram-positive
bacteria and other organisms. Some think this is
the oldest type of regulation because it can use
direct binding of metabolites and RNA.
25
Some systems using anti-terminator proteins
The protein family. They bind and block formation
of terminator hairpins.
The RNA targets (anti-terminators). The line
indicates nucleotides needed by the terminator
hairpin.
The EMBO Journal, Vol. 21, No. 8 pp. 1987-1997,
2002
26
The protein stabilizes base anti- forms
and blocks formation of a terminator
hairpin. Therefore it triggers transcription of
genes needed under these conditions.
27
Thats it. Questions about the exam?