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The regulatory roles and mechanism of
transcriptional pausing
R. Landick1
Department of Bacteriology, University of Wisconsin-Madison, Madison, WI 53705, U.S.A.
Abstract
The multisubunit RNAPs (RNA polymerases) found in all cellular life forms are remarkably conserved in
fundamental structure, in mechanism and in their susceptibility to sequence-dependent pausing during
transcription of DNA in the absence of elongation regulators. Recent studies of both prokaryotic and
eukaryotic transcription have yielded an increasing appreciation of the extent to which gene regulation is
accomplished during the elongation phase of transcription. Transcriptional pausing is a fundamental enzymatic mechanism that underlies many of these regulatory schemes. In some cases, pausing functions by
halting RNAP for times or at positions required for regulatory interactions. In other cases, pauses function
by making RNAP susceptible to premature termination of transcription unless the enzyme is modified by
elongation regulators that programme efficient gene expression. Pausing appears to occur by a two-tiered
mechanism in which an initial rearrangement of the enzyme’s active site interrupts active elongation and
puts RNAP in an elemental pause state from which additional rearrangements or regulator interactions can
create long-lived pauses. Recent findings from biochemical and single-molecule transcription experiments,
coupled with the invaluable availability of RNAP crystal structures, have produced attractive hypotheses to
explain the fundamental mechanism of pausing.
Introduction
Transcriptional pausing occurs when the normal catalytic
cycle of RNAP (RNA polymerase) becomes disrupted by one
or more structural rearrangements within the TEC (transcription elongation complex). All pausing characterized to
date is triggered by the interactions of nucleic acid strands
(RNA and DNA) with RNAP in the TEC or by auxiliary
regulators that contact RNAP, RNA, DNA or some combination of these TEC components. Thus pausing is programmed by nucleic acid sequence or by what can be
broadly termed pause signals. A fundamental property of
pausing is that only a fraction of transcribing RNAP molecules are affected by a given pause signal. Thus the pause
mechanism is branched. A fraction of RNAPs bypass the
site without pausing and a reciprocal fraction ‘recognize’
the pause signal and isomerize into the paused state. This
branched mechanism means that pausing at a given template
position or site can be characterized by two parameters: (i) an
efficiency of pause signal recognition, which is the fraction of transcribing RNAP molecules that enter the paused
state; and (ii) a pause duration, which is conveniently
expressed as a dwell time [reciprocal of the rate of pause escape
(ke )] or by a pause half-life (ln 2/ke ). Recognition of the
off-pathway nature of the paused state, reflecting a
branched mechanism between pause formation and pause
Key words: gene regulation, nucleotide addition cycle, pausing, RNA polymerase protein,
transcript elongation, transcription.
Abbreviations used: RNAP, RNA polymerase; TAR, transactivation-response element; TEC,
transcription elongation complex.
1
email [email protected]
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site bypass, has been reiterated in many studies from early
biochemical experiments [1,2] to contemporary single-molecule experiments [3–5]. Transcriptional pauses exhibit a wide
range of lifetimes, mechanisms and biological functions.
Biological function of transcriptional
pausing
In bacteria, transcriptional pausing plays a fundamental role
in coupling transcription and translation by halting RNAP
often to allow a translating ribosome to catch up to
RNAP and then release the enzyme from the paused state.
To maintain transcription–translation coupling, pauses need
only occur frequently enough to avoid exposing more than
100 nt of unstructured RNA, which is the minimal binding
site for the Rho transcription termination factor. Given that
most RNA exhibits significant secondary structure, even
low-efficiency pausing that occurs with sufficient frequency
will maintain coupling. Pausing also plays a key role as
the first step in both Rho-dependent and intrinsic (Rhoindependent) termination of transcription. Pausing halts
RNAP at terminators until Rho factor interacts with the
TEC and dissociates it via action of its ATP-dependent,
RNA helicase, or until a nascent RNA secondary structure
accomplishes the same task [6–9]. These pause signals often
encode U-rich RNA–DNA hybrids, which have the added
consequence of destabilizing the TEC [10]. The combined
effects of pausing in maintaining transcription–translocation
coupling and in facilitating Rho-dependent termination when
translation fails creates a prokaryotic version of an mRNA
Molecular Basis of Transcription
surveillance pathway [11], in which RNA damage or
mutation that would lead to defective protein synthesis causes
termination of transcription [12].
In bacteria, pausing also plays specific regulatory roles by
halting RNAP at key locations to allow interaction or recruitment of regulators. For instance, pause sites in the leader
regions of operons regulated by attenuation are thought to
halt RNAP to ensure that ribosomes are properly located
to control the termination decision. Pauses also appear critical to ensure binding of elongation regulators to RNAP, for
instance of λQ, to a promoter-proximally paused RNAP [13]
or of RfaH to RNAP paused at ops sites in the leader regions
of RfaH-regulated operons [14]. Additionally, specific
pause sites have been found to play important roles in the
proper folding of nascent RNA [15,16]. The recent discovery
of many additional attenuation mechanisms that are coupled
with small molecule–RNA interactions (riboswitches) highlights the importance of pausing to proper regulation, which
has been directly demonstrated for the FMN riboswitch [17].
In eukaryotes, the documentation of regulatory roles for
specific transcriptional pause sites has necessarily lagged
owing to the greater difficulty of in vitro experiments. Nonetheless, a variety of studies have suggested that pausing by
RNAPII may facilitate Tat-mediated regulation of HIV-I
transcription [18], facilitate polyadenylation [19,20], contribute to splice site selection in alternatively spliced mRNAs
[21,22], act synergistically with nucleosomes to create
barriers to RNA chain elongation [23], and make RNAPII
susceptible to abortive elongation (promoter-proximal premature termination) by factors such as NELF (negative
elongation factor) until appropriate phosphorylation patterns
are generated on the RNAPII C-terminal repeat domain by
kinases such as P-TEFb (positive transcription elongation
factor b) [24–26]. Elucidating the connections of pausing
by RNAPII to these regulatory events as well as possible
additional roles, for example in proper folding of RNA, is
an important objective for future studies of eukaryotic transcriptional regulation.
The structural basis of transcript
elongation
Understanding the fundamental mechanism of transcriptional pausing requires a complete knowledge of RNAP’s
nucleotide addition cycle as well as the accompanying structural changes in the TEC. The core structures of multisubunit
RNAP and the corresponding TEC are remarkably conserved
among all cellular organisms and consists mainly of two
large subunits (β and β or RPB1 and RPB2 in bacteria and
eukaryotes respectively) [27–30]. These two large subunits
make nearly all of the important nucleic acid contacts in
the TEC and form a deep cleft that surrounds RNA and
template DNA near the active site as well as side channels that
hold entering downstream DNA and exiting RNA transcript
(Figure 1). The large subunits are held together by two smaller
subunits (α 2 dimer or RPB3–RPB11 heterodimer in bacteria
and eukaryotes respectively), which bind on the backside of
the enzyme (not shown in Figure 1). Some contacts may
also be made by RPB5 to downstream DNA [30], by RPB7
to upstream RNA [31] in RNAPII, and by the C-terminal
domains of the α subunits to DNA or RNA in bacterial
RNAP. In addition to the main channel or cleft that holds
the 8–9 bp RNA–DNA hybrid, the key features of the
TEC include (i) a downstream DNA entry channel formed
between a trough in β and a lobe-like domain in β; (ii) a
melted transcription bubble of 12–15 bp; (iii) an RNA exit
channel that holds 4–6 nt of single-stranded RNA; (iv) a
secondary channel or pore that connects the active site to
the outside of RNAP and which is thought to function as the
NTP-entry channel, PPi exit route, or both; and (v) a bipartite
active site in which NTPs react with the 3 OH on RNA to
extend the RNA chain. The two subsites of the active site
have been variously called the i site or product (P) site
for the location that holds the RNA 3 -nucleotide and the
i + 1, substrate (S) site or A site for the location that holds
the substrate NTP (Figure 1A). An additional NTP-binding
site (the entry or E site) located downstream of the A site
may assist in NTP insertion into the A site, although the
location of this NTP pre-binding site is in dispute [32–35].
The main channel is bounded on the downstream side by
a long apparently flexible α-helix called the bridge helix,
which divides the cleft into the downstream DNA channel
and the secondary channel. Within the secondary channel, a
particularly flexible, yet highly conserved loop called the
trigger loop appears to be able to assume multiple conformations. Coupled movements of the trigger loop and bridge
helix have been postulated to play key roles in the nucleotide
addition cycle, for instance by assisting translocation or
nucleotide addition into the A site [36–39].
There are four fundamental steps in RNAP’s nucleotide
addition cycle (Figure 1B): translocation, NTP binding, catalysis, and pyrophosphate release. Pre-steady-state kinetic
analysis [34], single-molecule measurements of the forcedependence of transcription at low NTP concentration [40]
and structural modelling of the action of the transcription
inhibitor streptolydigin [38,39] all suggest that the processes
of NTP binding and translocation are coupled. The mechanism of this coupling is not yet established. Simple models
sometimes equated with a thermal ratchet suppose that NTP
binding in the A site traps an equilibrium between preand post-translocated states in the forward position [37,41].
Although this simple mechanism may well operate for singlesubunit RNAPs such as T7 [42], it is contradicted by
single-molecule measurements on multisubunit RNAPs [40],
which directly query the energetics of translocation. At least
two plausible alternatives exist [35]. NTPs bound in an E site
at the end of the secondary channel may destabilize the pretranslocated RNA 3 nucleotide as they attempt to rotate into
the A site, thus shifting RNAP to favour the post-translocated
conformation [38]. Alternatively, NTPs have been proposed
to bind to a different entry-site location in the downstream
DNA channel by base-pairing to the exposed portion of the
template DNA and thereby to facilitate translocation [34,43].
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Biochemical Society Transactions (2006) Volume 34, part 6
Figure 1 TEC structure and nucleotide addition cycle
(A) The structure of the TEC. Only the two large RNAP subunits are depicted. DNA strands are black; RNA is grey. The three
subsites (P, A and E) and other labelled RNAP features are described in the text. (B) The nucleotide addition cycle and
elemental pause mechanism. The four steps of nucleotide addition (NTP loading, translocation, catalysis and PPi release)
are depicted with the active site locations (P, A and E) from (A) highlighted. The elemental pause may reflect an inability to
couple NTP loading and translocation.
The mechanism of transcriptional pausing
Several lines of evidence suggest that transcriptional pausing
involves an initial rearrangement of RNAP into an off-line
state called the elemental pause in which nucleotide addition
is inhibited without a change of the translocation register of
RNAP. Single-molecules studies of transcription at high temporal resolution revealed that, even at saturating NTP concentrations, bacterial RNAP pauses frequently (once every
100–200 bp) for durations of 1–6 s on average [4,5]. These
so-called ubiquitous pauses are unaffected by applied force
that would either favour or oppose translocation [5], and
have been equated with the elemental pause state [44]. The
absence of force effects on ubiquitous pauses rules out
involvement of translocation events, such as backtracking or
hypertranslocation [45], in the formation of the elemental
pause state. Additionally, at least one well-defined ‘regulatory pause’ that occurs in the leader region of the his biosynthetic operon also has been shown biochemically to exist
in the pre-translocated register [46]. Thus the elemental pause
must form via an active-site rearrangement that does not
involve a change in translocation state. Likewise, escape from
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the pause is unlikely to be rate-limited by translocation,
since assisting or opposing forces do not decrease or increase
pause duration. One attractive possibility is that the elemental pause rearrangement compromises NTP-coupled
translocation (Figure 1B). If NTPs load into the active
site more efficiently when coupled with translocation, then
uncoupling this mechanism may make NTP loading more
difficult.
Longer-lived pauses are derived from the elemental pause
by one or more subsequent rearrangements or interactions
that further slow the rate of pause escape. Several different
types of rearrangements or interactions have been uncovered
(Figure 2). Although these different ways of stabilizing the
paused TEC have sometimes been used to define different
classes of pausing [45], it is important to recognize that in
some if not most cases two or more of these interactions
combine to prolong pausing. The most common way to prolong pausing involves reverse translocation (backtracking) by
RNAP on the nucleic acid scaffold [45,47–49]. Backtracking
is favoured when the nucleic acid scaffold is less stable in the
active translocation register than when RNAP backtracks to
create a new transcription bubble and RNA–DNA hybrid.
Molecular Basis of Transcription
Figure 2 Four ways to stabilize the paused state of the TEC
The four types of stabilized pauses described in the text are depicted. Examples where each type of mechanism has been
observed are given below each depiction.
The stability of the RNA–DNA hybrid is the dominant
contributor to this process. Upon backtracking, the RNA
3 -nucleotides displaced from the hybrid appear to enter the
secondary channel, where they may physically block NTP
entry. The backtrack pause mechanism has been characterized
for both bacterial RNAP and eukaryotic RNAPII. Careful
characterization of a backtrack pause in the early transcribed
region of HIV-1 revealed that it demarcates the template position at which the nascent RNA can rearrange into the TAR
(transactivation-response element) RNA secondary structure
[18]. Backtracking appears to inhibit TAR formation, whereas
forward tracking and pause escape are favoured when TAR
is formed.
At some pause sites, interaction of regulatory proteins can
prolong pausing. Two well-characterized examples in bacteria
are the promoter-proximal pause at the λ late promoter, which
is stabilized by an interaction of σ factor with the TEC [13],
and the ops pause, which is prolonged when RfaH binds the
paused TEC [14]. Interestingly, both σ and RfaH interact
with the single-stranded segment of the non-template strand
of the TEC, and both appear to stabilize RNAP in a backtrack
paused conformation. A third example of regulator-stabilized
pausing in bacteria involves the interaction of NusA with a
different class of paused TEC that is stabilized by formation
of a nascent RNA hairpin (hairpin-stabilized pausing) [45].
Hairpin-stabilized pausing may occur relatively infrequently
owing to the exact spacing requirements between the nascent
RNA hairpin and the location of an elemental pause site.
This spacing reflects the need for the pause hairpin to interact
with the so-called flap domain of RNAP [46]. Hairpinstabilized pauses are known to synchronize the transcription
of attenuation control regions with translation of leader
peptide coding regions in the leader regions of enterobacterial
amino-acid biosynthetic operons. The pause hairpin may act
as a trigger whose melting by the translating ribosome can
release the paused RNAP.
Finally, double-stranded DNA sequences that contact
RNAP downstream of the active site can profoundly in-
fluence transcriptional pausing [50]. The effect of these
sequences is not on the ease of duplex melting, but rather
appears to involve sequence or structure-specific interactions
with RNAP. The mechanism by which downstream sequences increase or decrease the longevity of pauses is not
well understood, but may involve effects on the movements
of the RNAP trigger loop.
Several important questions about the nucleotide addition
cycle and the mechanism of pausing remain to be answered.
Exactly how are the loading of NTPs into the RNAP active
site and the translocation of RNAP coupled? Do NTPs
indeed load via the secondary channel, or do they enter
through the alternative route of the downstream DNA channel in some or all cases? What are the exact physical rearrangements that occur in the elemental pause to disrupt NTPassisted translocation, and how are these rearrangements
stabilized by the various interactions that prolong transcriptional pausing. Answering these questions will provide the
fundamental framework to understand the mechanisms of
the myriad regulators that affect transcript elongation in both
prokaryotic and eukaryotic cells.
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Received 26 June 2006