Transcription
ISSN: 2154-1264 (Print) 2154-1272 (Online) Journal homepage: https://www.tandfonline.com/loi/ktrn20
Different types of pausing modes during
transcription initiation
Eitan Lerner, Antonino Ingargiola, Jookyung J. Lee, Sergei Borukhov, Xavier
Michalet & Shimon Weiss
To cite this article: Eitan Lerner, Antonino Ingargiola, Jookyung J. Lee, Sergei Borukhov, Xavier
Michalet & Shimon Weiss (2017) Different types of pausing modes during transcription initiation,
Transcription, 8:4, 242-253, DOI: 10.1080/21541264.2017.1308853
To link to this article: https://doi.org/10.1080/21541264.2017.1308853
Accepted author version posted online: 23
Mar 2017.
Published online: 27 Apr 2017.
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TRANSCRIPTION
2017, VOL. 8, NO. 4, 242–253
https://doi.org/10.1080/21541264.2017.1308853
POINT-OF-VIEW
Different types of pausing modes during transcription initiation
Eitan Lerner a, Antonino Ingargiola
and Shimon Weiss a,c,d
a
, Jookyung J. Lee
b
, Sergei Borukhov
b
, Xavier Michalet
a
,
a
Department of Chemistry & Biochemistry, University of California, Los Angeles, CA, USA; bRowan University School of Osteopathic Medicine,
Stratford, NJ, USA; cMolecular Biology Institute, University of California, Los Angeles, CA, USA; dDepartment of Physiology, University of
California, Los Angeles, CA, USA
ABSTRACT
ARTICLE HISTORY
In many cases, initiation is rate limiting to transcription. This due in part to the multiple cycles of
abortive transcription that delay promoter escape and the transition from initiation to
elongation. Pausing of transcription in initiation can further delay promoter escape. The
previously hypothesized pausing in initiation was confirmed by two recent studies from Duchi
et al.1 and from Lerner, Chung et al.2 In both studies, pausing is attributed to a lack of forward
translocation of the nascent transcript during initiation. However, the two works report on
different pausing mechanisms. Duchi et al. report on pausing that occurs during initiation
predominantly on-pathway of transcript synthesis. Lerner, Chung et al. report on pausing during
initiation as a result of RNAP backtracking, which is off-pathway to transcript synthesis. Here, we
discuss these studies, together with additional experimental results from single-molecule FRET
focusing on a specific distance within the transcription bubble. We show that the results of
these studies are complementary to each other and are consistent with a model involving two
types of pauses in initiation: a short-lived pause that occurs in the translocation of a 6-mer
nascent transcript and a long-lived pause that occurs as a result of 1–2 nucleotide backtracking
of a 7-mer transcript.
Received 10 February 2017
Revised 13 March 2017
Accepted 13 March 2017
Prolonged periods during which RNA polymerase
(RNAP) resides in a specific state can be used for efficient control and regulation of DNA transcription.
This drives us to study the slow processes that limit
the overall rate of transcription.
Transcription is generally divided into three major
stages: initiation, elongation and termination, where
initiation is usually the rate-limiting stage. Initiation
can be further divided into sub-stages.3,4 First, the
RNA polymerase (RNAP) holoenzyme is formed by
the association of the core enzyme (subunit composition a2,b,b0 ,v) with the specificity subunit s. Different
s factors recognize specific promoter sequences of
various groups of genes. The major s factor in Escherichia coli, s70, is responsible for recognition of
promoters for house-keeping genes. After association
with the RNAP holoenzyme, s70 binds to the
promoter DNA to form the RNAP promoter closed
complex (RPc), which involves binding at two
hexamer sequences centered around positions ¡10
CONTACT Shimon Weiss
90095-1569, USA.
© 2017 Taylor & Francis
sweiss@chem.ucla.edu
KEYWORDS
transcription initiation;
transcription pausing;
backtracking; abortive
initiation; promoter escape;
single-molecule FRET
and ¡35 relative to the C1 transcription start site.
After binding to the promoter, RNAP forms the
RNAP promoter open complex (RPo) through successive isomerization steps.5,6 During this process around
13 bp of DNA (from registers ¡11 to C2) must melt
to form the transcription bubble. The sequence of the
template strand of the transcription bubble serves as a
reading guide for the polymerization of an RNA transcript through base complementation. The two most
downstream DNA bases in the template strand of the
transcription bubble are coordinated with the active
site of RNAP, where the addition of NTPs into the 30
end of the nascent transcript occurs. The downstream
fork of the transcription bubble is located near the
active site, facing the RNAP secondary channel,
through which NTP substrates enter.7 The upstream
fork of the transcription bubble is located between
s70 regions 2 and 3. In initiation, the entrance to the
exit channel is blocked by the acidic tip of the s finger
domain (region 3.2, sR3.2), while the exit out of the
Department of Chemistry and Biochemistry, Box 951569, University of California, Los Angeles, CA
TRANSCRIPTION
channel is blocked by s region 4 (sR4). Structural and
biochemical data indicate that RNAP transition to
elongation requires a substantial spatial rearrangement of several RNAP domains, including the opening
of the b0 clamp and b flap,4,8 and the displacement of
sR3.2 and sR4 from the exit channel.9 It has been
suggested that this rearrangement leads to destabilization of s-promoter and s-RNAP interactions, thereby
facilitating transcript elongation and promoter
escape.10 Promoter escape is considered to be the
main rate-limiting step in initial transcription, after
RPo formation.
Initial transcript synthesis occurs via a DNA
scrunching mechanism, whereby downstream dsDNA
is reeled into the transcription bubble by promoterbound RNAP, enabling incorporation of NTPs into
the growing nascent transcript.11,12 DNA scrunching
leads to melting of dsDNA downstream to the bubble.
However, in initiation, the upstream fork of the bubble
is held tightly by interactions with sR2 and sR3. This
leads to a net increase in the size of the transcription
bubble in initiation, resulting in the formation of what
is better known as a “stressed intermediate.”13,14 In
this intermediate, each increase in bubble size is energetically compensated by an increase in the number of
transcript bases hybridized to the template strand.
Still, this complex is considered “stressed” due to the
additional space inside the RNAP–promoter complex
that has to be allocated for the DNA bases added into
the bubble by scrunching. As initial transcription proceeds, the nascent transcript grows longer with its 50
end approaching the negatively charged tip of the s
finger.15,16 The resulting clash is assumed to trigger
the removal of the exit channel blockage, inducing
promoter escape.10,15,17,18 Nevertheless, the stronger
the affinity of s to the promoter, the more probable
this encounter will not lead to the additional forwardtranslocation required to remove the s finger blockage
from the exit channel. Instead, this may lead to successive back-translocation of the downstream fork with
concomitant shrinking of the bubble and extrusion of
the short nascent RNA through the secondary channel, ultimately resulting in abortive transcript
release.19-22 After release, the complex reverts to the
RPo state for another attempt of RNA synthesis.14,2325
Multiple cycles of abortive transcription may occur
until the exit channel blockage is successfully
removed, enabling the transition to elongation and
the production of full RNA transcripts. Until recently,
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the conventional view was that promoter escape is a
rate-limiting step in transcription due to multiple
abortive initiation cycles, where each cycle of abortive
release could be rapid.14,26-28 However, experimental
data indicate that the release of abortive transcripts is
relatively slow, which may impede each single abortive
cycle.25,29 This calls for a thorough investigation of the
abortive release process.
The crystal structures of transcription initiation
complexes carrying a nascent transcript of 4–5 nucleotides,16 and up to 6 nucleotides in length30 have been
obtained. Structural analysis of the transcription bubble and the nascent transcript in these complexes
suggests that, starting from a 5-mer nascent transcript,
a steric clash forms between the acidic tip of the s finger and the phosphates of the 50 end of the nascent
transcript.16,31 The steric clash and the electrostatic
repulsion between the two negatively charged groups
grows larger the closer they get.10,15,17,18 Repulsive
forces may lead to the expulsion of the s finger,
elimination of the blockage of the entrance into the
RNA exit channel and may induce subsequent disruption of s-promoter interactions. Alternatively,
repulsion between the groups may push the nascent
transcript that leads to DNA anti-scrunching with
concomitant back-translocation of the nascent transcript and extrusion of the RNA 30 end into the
secondary channel. Successive back-translocation
steps ultimately lead to abortive release. However, in
each step of back-translocation, the distance between
the 50 end of the nascent transcript and the acidic tip
of the s finger increases, reducing the steric and electrostatic repulsions between the two groups. In the
presence of other stabilizing interactions, the backtracked transcript may be temporally stabilized in the
complex instead of being immediately released
through the secondary channel. The backtracked
nascent RNA may be stabilized, for instance, through
electrostatic attraction between the phosphate groups
of the extruded portion of nascent RNA and the
positively charged surface of the secondary channel. as
access of new NTPs into the active site is blocked by
the extruded RNA, this stabilization may lead to
transcriptional pausing. Additionally, structural data
suggests the non-electrostatic interactions between the
50 end nucleotide and acidic tip of the s finger may
actually contribute to stabilization of the nascent
transcript in the complex.16,30 Pausing in initiation
may also occur without backtracking. As the nascent
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E. LERNER ET AL.
transcript grows beyond the length of 6- or 7-mer, the
electrostatic and steric repulsion forces may also
impede the movement of the nascent transcript from
pre-translocated to post-translocated states.
Experimental evidence of pausing during
transcription initiation
In elongation, transcriptional pausing due to backtracking has been extensively characterized both in
vitro and in vivo.19,32-36 Pausing and backtracking
were hypothesized to occur in initiation, based on the
observations of initiation stalling,25,29 enhancement of
promoter clearance by GreA and GreB, and the susceptibility of abortive transcripts to cleavage by Gre
factors.9,14,18,37-44 Recently, using different experimental approaches, Duchi et al.1 and Lerner, Chung et al.2
have provided direct evidence of E. coli RNAP transcription pausing during initiation at a modified lac
promoter, lacCONS.45 Lerner, Chung et al. measured
the kinetics of single-round transcription reactions
with unlabeled reconstituted RNAP holoenzyme and
promoter DNA free in solution. They observed that
formation of a full-length transcript starting from a
late initiation stage (e.g., initially transcribing RNAP
promoter complex, ITC, synthesizing abortive transcripts of up to 7 nucleotides (nt) in length, RPITC 7)
was delayed as compared with transcription starting
from early initiation stages (order of delays: RPITC 7
> RPITC 6 > RPITC 4 > RPITC D 2). The average
delay in transcription kinetics was several minutes.
These results implied on transcriptional pausing, associated with certain states in initiation. At the lacCONS
promoter, transition to elongation occurs when the
transcript reaches a length of 11 nt;26,46,47 therefore,
RPITC 7, RPITC 6, RPITC 4 and RPITC D 2 states exist
during initiation. Control experiments showed that
the kinetics of a single-round run-off transcription
reaction starting from an early elongation complex
carrying an 11-mer transcript (RDE11) was almost
identical to that starting from an early initiation complex (RPITC D 2). One possible explanation for these
observations was that transcriptional pausing occurs
due to backtracking. Lerner, Chung et al. also found
that the transcriptional pausing reported above is
reduced in the presence of GreA. In elongation, transcript cleavage factors GreA and GreB catalyze hydrolysis of the 30 -terminal portion of the nascent
transcript that has backtracked into the secondary
channel.48-50 This action of Gre factors enables reassociation of the newly generated RNA 30 -terminus
with the active site, thus rescuing RNAP from the
paused-backtracked state and ultimately increasing
transcription activity. Gre factors act by inserting their
coiled-coil domain into the RNAP secondary channel.
Two acidic residues in the tip of the coiled-coil loop
stabilize the 30 end of the backtracked transcript and
coordinate the essential Mg2C ion at the RNAP active
site, catalyzing the endonucleolytic reaction. Under
the assumption that GreA acts by the same mechanism in initiation and in elongation, Lerner, Chung
et al. concluded that the observed delay in the synthesis of the full-length transcript is due to backtrackingassociated pausing during transcription initiation.
Moreover, the observed abortive transcripts produced
by RPITC 7 in the absence of GreA included 5-, 6and 7-mer RNAs, whereas abortive transcripts produced in the presence of GreA contained mostly a 6mer RNA and only trace amounts of 5-mer and 7mer RNAs. These results suggested that GreA promoted cleavage of 1–2 backtracked nucleotides. As
stated above, a stabilizing interaction between the 50 terminal nucleotide and the tip of the s finger has
been proposed based on the analysis of one of the
structures of the initiation complex.16 If backtracking
does occur, transcripts carrying a 50 OH group are
expected to back-translocate due to steric clash with
the s finger, while transcripts carrying a negatively
charged triphosphate group at the 50 end are expected
to be even further back-translocated due to electrostatic repulsion by the acidic tip of the s finger. Interestingly, Lerner, Chung et al. have shown that the
delay in transcription starting from RPITC 7 was longer when the 50 end of the transcript contained a triphosphate group (50 -priming of transcription with
pppApA), compared with when it contained an
uncharged OH group (50 -priming with ApA) (see
Supplementary information in Ref. 2). This observation suggested that the forces that induce backtracking are both steric and electrostatic. At the same
time, the contribution of the stabilizing interaction
between the tip of the s finger and the 50 -terminal
nucleotide of the nascent transcript (deduced from
the crystal structure16) may still occur but to a lesser
extent.
These experiments were performed using a partial
set of NTPs that only allowed formation of RPITC 7. It
was crucial to examine the phenomenon of pausing in
TRANSCRIPTION
initiation with all four NTPs at concentrations comparable to physiologic NTP pools (under standard conditions, 0.1–1.0 mM).51 In addition, the abovementioned
experiments provided an indirect evidence for pausing
due to backtracking in transcription initiation.
Single-molecule experiments offer direct observation of biochemical events with individual molecules.
To follow the actual progression of transcription complex from initiation to elongation, Lerner, Chung et al.
tracked the size of the transcription bubble of individual DNA molecules in real-time. They used a magnetic tweezers assay, which applies tension on and
tracks the height of a magnetic bead attached to a surface-tethered DNA carrying the lacCONS promoter
sequence. In this assay, the dsDNA is negatively
supercoiled to a given level by rotating the magnetic
bead. This supercoiling forms plectonemes in the
DNA. In this mode of supercoiling, melting of base
pairs induces an increase in the number of plectonemes, lowering in the height of the bead. Therefore,
the height of the bead is a direct measure of the number of melted bases involved in the transcription bubble. When DNA is free and not bound to RNAP, no
DNA bubble is formed, setting a maximum bead
height. Bubble opening during transcription lowers
the bead height and upon initiation, DNA scrunching
leads to increase in bubble size and further lowers
bead height. Unlike in initiation, the bubble size is
minimal in elongation and bead height increases (but
not up to the level of an unbound DNA) for a short
time, because elongation is fast.52-55 If backtracking
occurs during initiation, back-translocation leads to
anti-scrunching, which in turn leads to decrease in
bubble size. This should lead to a decrease in bubble
size relative to scrunched bubble size in initiation but
not as small a bubble size as in elongation, leading to a
bead higher than in scrunching in initiation but lower
than in elongation. Additionally, if the backtracked
state is associated with pausing, dwell time in this state
is expected to be longer than in the scrunched initiation state. This assay allowed Lerner, Chung et al. to
observe traces of bead height as a function of time,
corresponding to states of the DNA bubble during a
complete transcription cycle from free DNA, to initiation, elongation, and back to free DNA. When GreA
was present, initiation-associated dwells were shortlived (350 § 30 s) and had a minimal bead height
compatible with maximal bubble size (16.2 § 0.2 bp).
However, in the absence of GreA, a nonnegligible
245
portion of initiation-associated dwells was long-lived
(4,600 § 2,700 s) and had transcription bubbles with
decreased sizes (80% with 14.8 § 0.4 bp and 20% with
9.9 § 0.3 bp). These results suggested direct evidence
of pausing in transcription initiation associated with
backtracking.
Although magnetic tweezer transcription assays
allow the observation of full transcription cycles at the
single-molecule level for long periods of times
(»10,000 s per molecule on average), they require
molecule-by-molecule measurement, which makes
accumulating statistics from many single molecules
time consuming. In addition, the time resolution for
the observation of bubble size-associated transitions is
1 s (average of 31 Hz), which precludes fast kinetic
analysis. An alternative method of following transcription of single molecules in real-time by changes in the
bubble size is by monitoring changes in FRET efficiency between two fluorescent dyes, placed at positions upstream and downstream from the bubble.
Duchi et al. performed such experiments and their
results are quite intriguing. Upon supplying the partial
set of NTPs that allows reaching RPITC 7, they
observed transcriptional pausing that lasts on average
20 s in a state where the size of the bubble corresponds
to a scrunched initiation complex carrying a 6-mer
transcript (RPITC D 6). The size of this bubble can also
correspond to a complex carrying a 7-mer transcript
(RPITC D 7) after one nucleotide backtracking. In both
states, six DNA bases are scrunched into the bubble
(RPbubble D 6). In some of the FRET traces, Duchi et al.
observed long dwell times in RPbubble D 6 following
short excursions of 5 s on average into RPbubble D 7,
which the authors referred to as pausing after backtracking. In single-molecule FRET (smFRET), one of
the main limitations is that single-molecule observation times are limited by fluorophore photobleaching.
In cases of states with dwell times longer than the
available observation time, this may lead to observation of FRET states without a characterized dwell
time. Some of the traces were long enough (400 s) to
suggest pausing at RPbubble D 6 for times longer times
(> 400 s). While 55% of these long smFRET traces
have shown both fast (»8 s) and slow (»55 s) cycling
between the paused state in RPbubble D 6 and RPo, 45%
of these FRET traces show pausing in RPbubble D 6 for
periods that encompass the whole observation time.
Overall, these results were interpreted by Duchi et al.
as events that were either on-pathway, or off-pathway
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E. LERNER ET AL.
of transcript synthesis, as part of backtracking and
abortive release. However, in this model, the shorter
lived paused state in RPITC D 6 on-pathway of transcript synthesis (»20 s) was viewed as rate limiting.
This interpretation was justified due to the majority of
FRET traces showing pausing at RPbubble D 6 without
first reaching RPbubble D 7; hence, it served as a plausible explanation given the experimental results. In
addition, the authors explained that the long-lived
RPbubble D 6 paused states could not represent backtracked states since the long-lived paused RPbubble D 6
state started without prior excursions to RPbubble D 7,
required for pausing after backtracking. Yet, it may
well be that such prior excursions to RPbubble D 7
before backtracking to RPbubble D 6 occur faster than
the reported time resolution (integration into bins of
200 ms). In the magnetic tweezers experiments, with a
time resolution of 1 s, Lerner, Chung et al. also
observed bubble size changes from no bubble in free
DNA directly to its scrunched form in initiation. The
pausing times observed with magnetic tweezers transcription assays (4,600 § 2,700 s) were much longer
than typical single-molecule trajectory observation
times (a few hundreds of seconds) and may be the
extension of the stably paused state reported by Duchi
et al. The magnetic tweezer experiments of Lerner,
Chung et al. showed clear GreA dependence of both
dwell times and bubble sizes in initiation-associated
dwells. Thus, repeating these experiments in the presence of GreA may help validate the interpretation proposed by Duchi et al. If the paused state of RPbubble D 6
corresponds to RPITC D 6, with no backtracking, GreA
will not affect its dwell time. Alternatively, if RPbubble
D 6 corresponds to RPITC D 7, with one nucleotide
backtracked, then GreA should induce transcript
cleavage, thereby decreasing the dwell time of the
paused state.
Additional experimental evidence showing the
decrease in bubble size in the pausedbacktracked initiation intermediate
To show that pausing in initiation can be rate-limiting
during promoter escape, and to discriminate this
Figure 1. Promoter bubble opening activity (POA) and promoter escape activity (PEA) of doubly labeled lacCONS promoter dsDNA. Each
panel reports the proximity ratio histogram for FRET between donor (D; ATTO550 or Cy3B in the case of (C15)TA¡(¡15)NTD) and
acceptor (A) labeling different promoter registers on either the template (T) or the nontemplate (NT) strands. From left to right, two
panels report results for both dyes in the bubble region in initiation, the third panel reports results when one dye was upstream from
the bubble region in initiation and the other was downstream from it, and the last two panels report results when one dye was in the
bubble region in initiation, while the other downstream from it. From top to bottom, shown are proximity ratios of the free promoter
DNA, in within the RPITC D 2 initiation complex and after run-off should have occurred following supply of all four NTPs. f, g and h represent the fraction of the sub-population that is not the major one in the measurement of free promoter DNA and their values are
reported as a result of a constrained two Gaussian fit (see the appendix). Promoter bubble opening activity (POA) and promoter escape
activity (PEA) are calculated from these fractions as described in the appendix.
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247
Figure 2. (A) Proximity ratio histograms exhibit a sub-population with decreased transcription bubble size in RPITC 7, which is reversible
by addition of increasing amounts of GreA. Proximity ratio histograms of (¡8)TA¡(¡5)NTD are shown. From top to bottom, histograms
are shown for the conditions indicated in the labels of each panel. The solid black vertical line denotes the peak PR of a sub-population
whereby the DNA between the dyes is annealed (either free promoter DNA or RNA-promoter closed complex, see vignettes). The
dashed black vertical line denotes the peak proximity ratio the melted DNA in the open bubble sub-population in RPITC D 2 (see
vignette). The dashed red vertical line denotes the peak proximity ratio of a sub-population that is intermediate between the sub-populations of closed and opened bubble DNA. Sample preparation, measurement and analysis performed as explained by Ingargiola
et al.,59 only with triphosphate adenylyl(30 –50 ) adenosine (pppApA, generously provided as a gift by Prof. Richard Ebright), instead of
adenylyl(30 –50 ) adenosine (ApA). Sub-population fitting analysis was performed as described in the appendix. (B) Two different paused
initiation states as described by Duchi et al.1 and by Lerner, Chung et al.,2 and a model that combines both types of initiation pausing.
RPITC D 6, pre and RPITC D 6, post denote RPITC D 6 with the 30 -terminal base positioned at the pre- and post-translocated sites in RNAP,
respectively. RPITC D 7, pre is defined in the same way as RPITC D 6, pre, but for a 7-mer nascent transcript. RPITC D 7, 1 bt denotes an initiation
complex with a 7-mer nascent transcript backtracked by one base.
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E. LERNER ET AL.
pausing from events related to inactive (moribund)
initiation complexes,56 we performed microsecond
alternating laser excitation (msALEX) smFRET measurements57,58 using doubly labeled lacCONS promoter
DNAs that are freely diffusing in a confocal setup. In
this type of smFRET measurement, the proximity
ratio (PR; FRET efficiency uncorrected for leakage,
direct excitation and g factor, see the Appendix) is
reported for individual molecules transiting through
the detection volume during very short bursts (typically ms). The PR values of many different single-molecule bursts are accumulated in histograms in which
only abundant and stable states are represented as distinct sub-populations. Similar to the approach taken
by Duchi et al., we also tracked the PR between the
dyes placed at various positions around the transcription bubble. The labeled promoter DNA used for these
experiments were chosen based on the following criteria: (i) exhibiting significant change in PR upon bubble opening; (ii) exhibiting significant promoter
bubble opening activity (POA) upon incubation of
RNAP; and (iii) exhibiting significant promoter escape
activity (PEA) upon incubation with all NTPs (see the
appendix). We tested five doubly labeled lacCONS
promoter constructs for these criteria and the results
are summarized in Figure 1. For the sake of clarity, we
use the following nomenclature to indicate a doubly
labeled promoter, (C10)TA¡(¡5)NTD is a construct
with an acceptor dye (ATTO647N) on a template base
at register (C10) and with a donor dye (ATTO550 or
Cy3B) on a nontemplate base at register (¡5). In the
case of (C10)TA¡(¡5)NTD and (¡8)TA¡(C10)
NTD, the change in PR due to bubble opening was
expected to be minuscule as expected from assessment
of the crystal structure,30 and indeed the differences
between their PR histograms in free DNA, RPo and
run-off conditions (after addition of all NTPs) are
small. Fitting the PR histograms to constrained double
Gaussians yielded POA and PEA values that were
either small or negligible. One construct that is
expected to exhibit a large increase in PR moving
from free DNA to RPITC D 2 is a construct in which
one dye is upstream and another is downstream from
the bubble region, such as in (C15)TA¡(¡15)NTD
(donor Cy3B). This construct exhibits a sub-population with increased PR values compared with that of
the free DNA sub-population. Yet, POA and PEA are
small. Interestingly, during initiation the constructs
with both dyes in the bubble region, (¡8)TA¡(¡5)
NTD and (¡8)TA¡(¡3)NTD (donor ATTO550)
exhibit lower PR values compared with the free promoter sub-population, due to increase in the size of
the bubble and plausible activities (> 45% POA and
PEA), especially with respect to the (¡8)TA¡(¡5)
NTD construct. We therefore chose to report initiation effects using the construct that exhibits substantial POA (0.71 § 0.02) and PEA (0.71 § 0.05) values
that are the largest among the set of doubly labeled
promoter constructs tested. It is important to mention
that the kinetics of transcription starting from RPITC
D 2, measured by the single round quenched kinetics
assay,2 was very similar to the promoter escape kinetics measured by Ingargiola et al. with an 8£1 multispot smFRET setup, following the open bubble subpopulation in (¡8)TA¡(¡5)NTD after addition of all
NTPs.59 This indicates that promoter escape kinetics
of RPITC D 2 with dyes in the bubble region follow similar run-off kinetics for unlabeled DNA.
We then compared the PR histograms of the initiation complex in different states (Fig. 2A) including:
RPITC D 2 (RPo in the presence of 500 mM pppApA),
RPITC 4 (RPo in the presence of 500 mM pppApA,
100 mM UTP), RPITC 7 (RPo in the presence of 500mM pppApA, 100 mM UTP and GTP) and RPITC 7
in the presence of increasing concentrations of GreA
(2, 5, 10 and 20 mM). The solid black vertical line on
the right denotes the peak of high PR, corresponding
to the sub-population of free promoter DNA or RPc
state. The peak PR value of this sub-population is not
expected to change dramatically. The dashed black vertical line denotes the peak of the center value of PR for
the open transcription bubble sub-population in
RPITC D 2. The dashed red vertical line denotes the
peak of the center value of PR for any additional PR
sub-population that may appear as a result of scrunching, backtracking, or response to GreA binding. In general the bubble size changes due to DNA scrunching,
but it is not clear whether this change will be reflected
in PR values of the open transcription bubble sub-population for this construct (¡8)TA¡(¡5)NTD. The
crystal structures of initiation complexes corresponding
to different steps in initiation show only minor changes
in the distance between DNA bases at registers (¡8)
and (¡5) of the template and nontemplate strands,
respectively.16,60 Considering the bulky size of the dyes
(including the linkers), the distance between the dyes
attached to these bases may not show substantial
change in PR upon scrunching. The PR histogram for
TRANSCRIPTION
RPITC D 2 includes two sub-populations with peak PR
values 0.90 § 0.02 and 0.51 § 0.03 of DNA with
closed and opened bubble, respectively. These two PR
sub-populations also appear in all other measured initiation states, namely in RPITC 4, RPITC 7 in the
absence and presence of GreA (Fig. 2A, solid and
dashed vertical black lines). Upon moving from RPITC
D 2 to RPITC 4, we identify an additional sub-population with a peak PR value that is intermediate between
the values of the closed and opened DNA bubble (0.77
§ 0.04; Fig. 2A, vertical red line). However, in RPITC
4, the contribution of this additional sub-population to
the overall PR histogram is small (5%). It is only in
RPITC 7 that the contribution of this intermediate
sub-population is maximal (24%). This may indicate a
large portion of initiation complexes with reduced bubble sizes that are abundant and long-lived enough to be
represented in a PR histogram of freely diffusing single
molecules. This finding is in agreement with both
Lerner, Chung et al. magnetic tweezers findings and
smFRET studies of immobilized initiation complexes
published by Duchi et al. Subsequently, we tested the
response of the open bubble PR sub-population in
RPITC 7 as a function of increasing concentrations of
GreA. The results show that GreA decreases the contribution of the intermediate PR sub-population to the
overall PR histogram close to the levels observed in
RPITC D 2 (down to 3% in 20 mM GreA), as if no
change in the bubble size had occurred. We interpret
these results to indicate that pausing in initiation occurs
due to backtracking, which causes reduction in bubble
size, and which is reversed by the action of GreA.
Conclusions – a unified model of pausing in
transcription initiation
Our experimental results complement those reported
by Duchi et al. We propose that there are two types of
pausing in initiation at the lacCONS promoter. One is
short-lived (»20 s) and involves formation of RPITC D
0
6 (with RPbubble D 6) with the nascent transcript 3 end
in the pre-translocated position (RPITC D 6, pre), as suggested by Duchi et al. Another pausing is long-lived
(thousands of seconds) and involves formation of
RPITC D 7 (with RPbubble D 6) with nascent RNA backtracked by one nucleotide (Fig. 2B). As multiple
occurrences of a short-lived pausing would be
equivalent to a few long-lived pauses, both pauses
may contribute to the rate-limiting nature of promoter
249
escape. An experiment demonstrating that GreA
functions during initiation the same way that it does
during pausing and backtracking in elongation would
support the mechanism we proposed here.
Disclosure of potential conflicts of interest
No potential conflicts of interest were disclosed.
Acknowledgments
We thank Dr. SangYoon Chung for fruitful discussions, Ms.
Maya Segal for language editing and Mrs. Maya Lerner for
preparation of illustrative vignettes of RNAP and promoter
DNA, and also Prof. Richard Ebright for generously providing
us with the triphosphate dinucleotide pppApA, as a gift. SW
discloses equity in Nesher Technologies and intellectual property used in the research reported here. The work at UCLA
was conducted in Dr. Weiss’s Laboratory. The content is solely
the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Funding
This work was supported by the NIH under grant numbers
GM069709 (to SW) and GM095904 (to XM and SW); NSF
under grant number MCB-1244175 (to SW).
Authors’ contributions
EL and SW designed this study; EL developed, designed and
performed the smFRET measurements and analyzed the data,
in consultation with AI and X.M; JJL and SB prepared GreA
protein; EL, SB, XM and SW wrote the point of view.
ORCID
Eitan Lerner
http://orcid.org/0000-0002-3791-5277
Antonino Ingargiola
http://orcid.org/0000-0002-9348-1397
Jookyung J. Lee
http://orcid.org/0000-0002-6551-2870
Sergei Borukhov
http://orcid.org/0000-0002-3517-3003
Xavier Michalet
http://orcid.org/0000-0001-6602-7693
Shimon Weiss
http://orcid.org/0000-0002-0720-5426
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Appendix
In single-molecule msALEX analysis, three streams of
photons are analyzed: donor and acceptor fluorescence
photons during green laser excitation (noted here as
DD and DA, respectively), and acceptor photons during red laser excitation (noted here as AA). Burst photon counts in each of these photon streams, are
background-corrected by subtracting the burst duration times the background rate. A dual channel burst
search (DCBS; intersection of bursts from green excitation burst search and red excitation burst search)61 is
performed using m D 10 and F D 6, to isolate the FRET
data from non-FRET data (Donor-Only and Acceptor
Only) for further analysis. Next, the following two conditions are used to filter smFRET burst data:
1. nDD C nDA 50
2. nAA 30
where nDD, nDA and nAA are the number of photons
in a burst in streams DD, DA and AA, respectively,
corrected for background counts. Then, proximity
ratio, PR, is calculated for each burst:
nDA
PR D
nDD C nDA
PR is related to FRET efficiency; however, it is not corrected for leakage of donor fluorescence into the
acceptor detection channel (lk), direct excitation of
the acceptor with the laser intended for donor excitation (dir) and for the ratio of donor acceptor fluorescence quantum yields and the ratio of detection
efficiencies of the 2 detection channels (g factor).
We assume the values of these correction values are
similar for a given doubly labeled DNA construct
across different states. We compare relative results;
thus, we report on PR and not on FRET efficiency.
To calculate the promoter bubble opening activity
(POA) and the promoter escape activity (PEA), PR
histograms of doubly labeled promoter in its free
dsDNA (free promoter) form, RPITC D 2 and run-off
forms were fitted to two Gaussian functions. PR
sub-populations can be approximated by a Gaussian
function. Nevertheless, the quality of Gaussian fits,
being functions with no skewness, is affected by the
skewness of the distribution toward peak PR value of
0.5. The farther the peak PR value is from 0.5, the
larger this skewness is. We approximated the PR
TRANSCRIPTION
histograms with fitting of two Gaussians, where one
Gaussian described the sub-population of promoter
DNA that is free of binding to RNAP. The second
sub-population represented the conformation of the
open transcription bubble. However, due to the skewness of PR sub-populations toward a peak value of 0.5,
it is possible that some spurious bursts with peak PR
values outside the free promoter sub-population may
also contribute to the second sub-population. For this
reason, we constrained the fitting to two Gaussians
with the major PR sub-population in free promoter,
and we used the area under the second PR sub-population to quantify ratiometric quantities related to
bubble opening activity and bubble closure upon promoter escape activity. We defined f, g and h as the
fraction of the histogram that is occupied by the subpopulation with center PR values. f for free promoter,
g for RPITC D 2 and h for run-off. We defined two
quantities:
1. Promoter bubble opening activity: POA
D1¡
2. Promoter escape activity: PEA D
f
g
1 ¡ hg
1¡f
The definition of POA accounts for the relative
increase in the fraction of bursts in the second PR
sub-population, due to addition of initiation complexes with opened transcription bubble. The definition of PEA accounts for the relative decrease in
the fraction of bursts in the second PR subpopulation, due to bubble movement to downstream DNA, which leads to DNA re-annealing of
the bubble region in initiation. This definition
253
also takes into account that some bursts in the
second PR sub-population are spurious and seen in
measurement of the free promoter. The error
estimation of these activity values are propagated,
using these equations, from the fitting errors on f,
g and h.
To quantify the presence of PR sub-populations
and their contributions to the PR histograms in
Figure 2A, we fitted a function of sum of Gaussians to
the PR histograms. As in Figure 1, we used a sum of
two Gaussians to fit the PR histograms of doubly
labeled promoter in its free dsDNA (free promoter)
form, RPITC D 2 and run-off forms. Then, we used a
sum of three Gaussians to fit the PR histograms of
RPITC 4, RPITC 7 in the absence and presence of
GreA, where the peak PR values of two sub-populations were constrained to the values of the two subpopulations in the fitting results of RPITC D 2. This
allowed elucidating a third sub-population and its relative contribution.
Data analysis and figure preparation was performed
using FRETBursts, an open source burst analysis program for smFRET data.62 Python notebooks that
describe the data analysis from raw data to the results
presented in the figures are available in https://fig
share.com/s/2b3399ace2a6a9b2bcec (DOI: 10.6084/
m9.figshare.4590721). Raw data is saved in PhotonHDF5 format63 and is available in https://figshare.com/s/
489f12461399cc2adca4 (DOI: 10.6084/m9.figshare.4588
741) and in https://figshare.com/s/87ad049f556d189
66a80 (DOI: 10.6084/m9.figshare.4589560).