Promoter escape, pausing, and termination
R. Landick. September 29, 2004
Figure 1. Abortive Initiation
amount and pattern of abortive
A The
transcription varies widely
Page 1
B. Model of promoter escape. After Hsu
2002 Biochim Biophys Acta 1577:191,and
transcription complex depictions of
Murakami & Darst 2003 Curr Opin Struct
Biol 13:31
Open Complex
Initiation of RNA
Synthesis
Run off
transcripts
Abortive
Initiation
Extruded
loops of
scrunched
DNA strands
-35
-10
Stressed
Intermediate
Abortive
transcripts
Energy
From Hsu et al. 2003 Biochemistry 42:3777,
Fig. 2. The pattern of of transcripts produced
from three promoters during synchronous
transcription started with all 4 NTPs and
gamma-32PATP label. The runoff for T7 A1
and N25 is 50 nt and for N25 anti is 64 nt.
TEC
Promoter/σ
Promoter/σ
C Initial synthesis
Stressed
RNA
release
release
Intermediate release
Hybrid
bp raise
Ge
RNA
release Ga
release
barrier
Ge
DNA scrunching
Ga
destabilizes
complex
Open
Initiating
Open
Initiating
TEC
TEC
Complex
Complex
Complex
Complex
abort
escape
abort
escape
The process of abortive initiation is most easily appreciated by considering the
energetics of the complex during promoter escape. As RNAP makes the initial transcript,
scrunching (DNA strands looping out) destabilizes the complex and makes escape
easier, whereas the growing RNA:DNA hybrid raises the barrier to abortive transcript
release. What predominates at a particular promoter depends on the energetics of
these different contirbutions. (The case illustrated is perhaps closest to the N25 promoter).
Promoter escape, pausing, and termination
R. Landick. September 29, 2004
Page 2
Figure 2.. Many factors contribute to the regulation of promoter escape. See (Hsu
2002 Biochim Biophys Acta 1577:191; Hsu et al. 2003 Biochemistry 42:3777; Vo et al.
2003 Biochemistry 42:3798; Vo et al. 2003
Biochemistry 42:3787)
promoter contacts can restrain RNAP (eg,
A Strong
Ellinger et al. 1994 J Mol Biol 239:466).
B
Strong interactions of σ with RNAP can inhibit
escape. The sigma region 3.2 loop, which passes
near the active site and lies in the RNA exit channel,
appears to promote abortive transcription (and
therefore inhibit promoter escape (Murakami et al.
2002 Science 296:1285).
C Extruded DNA sequences will affect stability
of the stressed intermediate, depending on
how many bp scrunch, how GC-rich the
sequence is and whether the loops can form
and structure (or interact with regulators?).
D
Initially transcribed sequence can favor or disfavor
backtracking or abortive release, depending on the
stability of the hybrid possibly interactions with
RNAP.
E
DNA-binding transcription factors can favor or disfavor
escape. Examples: Phage Phi29 P4 protein inhibits
escape through cooperative binding interactions with the
alpha CTD (Monsalve et al. 1996 EMBO J. 15:383). Phage
P22 Arc protein promotes escape by competing for promoter contacts (Smith & Sauer 1996 Proc. Natl. Acad. Sci.
USA 93:8868).
F
Gre factors can promote escape either by cleaving the
initial RNA to favor forward transcription or by inhibiting
release out the secondary channel (Feng et al. 1994 J.
Biol. Chem. 269:22282; Hsu et al. 1995 Proc. Natl. Acad.
Sci. USA 92:11588)
Gre
Promoter escape, pausing, and termination
R. Landick. September 29, 2004
Page 3
Figure 3.. Biological roles of transcriptional pausing. Examples of each of these
have been described. A. The basic paradigm of pausing.
A
Unassisted, RNAPs pause
ubiquitously with varying
efficiency and duration
B
Promoter-proximal
pausing can regulate
productive initiation
C
Misincorporation can trigger
pausing; RNA cleavage
then relieves pausing.
D
Pausing can expose
binding sites and
synchronize regulatory
molecule recruitment
E
Pausing couples transcription
to ribosome movement or
spliceosome deposition.
correct
folding
F
versus
misfolding
G
Pausing can facilitate
proper RNA folding
Pausing creates
intermediates
leading to termination
Figure 4.. RNA structure, backtracking, or regulatory protein interaction can increase
the lifetime of the paused state.
NTP
Hairpin
Stabilized
Backtrack
Stabilized
Regulatory
Molecule
Promoter escape, pausing, and termination
R. Landick. September 29, 2004
Page 4
Figure 5.. Mechanistic dissection of one type of
paused TEC, the hairpin stabilized TEC, using population averaged kinetic studies. (see Chan et al.
Clamp
1997 J. Mol. Biol. 268:54; Artsimovitch & Landick
1998 Genes Dev, 12:3110). This pause event is triggered by sequences in the hybrid, active site, and
downstream DNA, and prolonged by interactions of
Flap
all three of these regions plus that of the pause hairNTP
pin. The contributions of all 4 pause signal components to pause duration are additive. This pause signal plays role E in Fig. 3 at a specific site in the
leader region of the his operon. Interaction of the
Pause Hybrid Active- Downstream
ribosome with the hairpin released the paused TEC.
Hairpin
DNA
Note the key spacing of the hairpin from the 3' end
site Nt
(11 nt), which places the hairpin in the exit channel
without disrupting the hybrid.
Bypass
0
time
N
N-1
RO( )
N+1
Isomerize
[RNA]
Escape
Pause
1
1
Efficiency=fraction TECs
that pause
t1/2=half-life for
escape
Efficiency= 0.8
0.8
0.6
P( )
0.4
0.1
0.2
100
200
time (s)
0
t1/2= 52 s
100
0
200
GU
C
A
U Pause
U
Multipartite his C
U Hairpin
pause signal
A
U
C
G
A
U
RNA::DNA active
downstream
antisense oligo
site
G
C
hybrid
DNA
UCAUCACCAUCAU C
G CG AUGUGUGCU GGAAGACATTCA
A29
P
U-less
T7 A1 Promoter
1
his pause
A29
antisense oligo
RO
hybrid region mutant
downstream DNA mutant
t1/2= 52 s E=0.8
0.1
0
t1/2= 6 s E=0.7
10
20
30
0
t1/2= 12 s E=0.1
t1/2= 29 s E=0.3
10
0
20
30
10
20
30
Promoter escape, pausing, and termination
R. Landick. September 29, 2004
Page 5
Figure 6.. Single-molecule assay of transcriptional pausing. See Wang et al. 1998 Science
282:902; Neuman et al. 2003 Cell 115:437; Shaevitz et al. 2003 Nature 426:684).
A.. Pausing occurs often, but only infrequently for significant lifetimes. Different RNAP molecules
exhibit different pause profiles, as expected for variable efficiency pausing for varying lifetimes.
2nd RNAP
(offset)
B.. The ubiquitous pauses are not affected by applied force.
C.. The longer pauses are affected by force and, for many, backtracking can actually be detected.
600
Time spent in long pauses/kb (s)
500
+1 mM ITP
400
300
200
+1 mM ITP
+Gre factors
100
+Gre
factors
+1 mM ITP
+Assisting force
Promoter escape, pausing, and termination
R. Landick. September 29, 2004
Page 6
Figure 7.. Possible model of transcriptional pausing. An initial rearrangement leads to
inhibition of NTP binding in the active site. One idea is depicted here, involving placement
of the trigger loop to block the E site. Other movements, including of the 3 base cannot
be excluded. Subsequent backtracking or hairpin interaction could stabilize the paused
state. Regulatory molecules either could further stabilize the backtracked or hairpin
paused TECs, or could directly stabilize the transient paused state.
BEFORE
SITE
tight
NTP binding
weak
binding
TRANSIENT
PAUSED STATE
ktr
bac
ack
STABILIZED (LONG-LIVED)
PAUSED STATE
(more backtracking)
ARREST
AFTER
SITE
Promoter escape, pausing, and termination
R. Landick. September 29, 2004
Page 7
Figure 8.. Intrinsic termination signals.
Like the pause signal, an intrinsic terminator is multipartite, but the hairpin reaches
to within 8 of the site of termination.
Many terminators contain a perfect U-tract for the hybrid, which greatly destabilizes
it. rU-dA bp contribute only 0.2 Kcal/mol to the TEC, whereas all other bp are 1.7
to ~3 Kcal/mol (rA-dU and GC bp, respectively).
The two absolutely conserved features of intrinsic terminators are the spacing between
the hairpin and the termination site and the presence of at least 3 Us immediately
after the hairpin.
A.. Examples of termination signals.
A
A
U
C
C
G
C
C
C
G
AUCAGAUACCC A
U
trp terminator
G
A
G
C
G
G
G
C Hybrid
U UUUUUUU GAACAAAATTAG
Terminator
Hairpin
Downstream
DNA
U
U
C
C
A
C
U
C
G
UGUAAUCACACU G
C
T7 early terminator
G
G
G
U
G
G
G
Hybrid
C
C UUUCUGCG TTTATAAGGAGA
Terminator
Hairpin
Downstream
DNA
BYPASS
B.. Possible mechanisms of intrinsic termination. The actual mechanism is unresolved.
A recent publication argues for the Pull-out mechanism (Santangelo & Roberts 2004 Mol Cell
14:117).
BEFORE
SITE
AFTER
SITE
Hairpin pulls RNA
out the exit channel
Note, this requires
forward translocation
unless a perfect
U-tract is present.
Allosteric effect
of hairpin
disrupts hybrid
Hairpin
Invasion
RNA
Pull-out
Promoter escape, pausing, and termination
R. Landick. September 29, 2004
Page 8
Figure 9.. Rho-dependent termination signal.
A. Structure of Rho (Skordalakes & Berger 2003 Cell 114:135).
N-terminal
RNA-binding
domain
C-terminal
ATPase
domain
B. Mechanism of Rho action. Rho prefers to bind unstructured RNA, and apparently loads
through the cleft in the hexamer. Binding can be greatly stimulated by certain poorly defined
sequence elements called rho-utilization or rut sites. Rho is an ATP-dependent RNA helicase,
and translocates to reach RNAP once it loads.
Paused TEC
Ribosome releases if mRNA is
correct so translation occurs
If no ribosome (mRNA defective)
or not an open reading frame,
Rho terminates RNAP.
ρ