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σ70-dependent Transcription Pausing in Escherichia coli
doi:10.1016/j.jmb.2011.02.011
J. Mol. Biol. (2011) 412, 782–792 Contents lists available at www.sciencedirect.com
Journal of Molecular Biology j o u r n a l h o m e p a g e : h t t p : / / e e s . e l s e v i e r. c o m . j m b
REVIEW
σ 70 -dependent Transcription Pausing in Escherichia coli Sarah A. Perdue and Jeffrey W. Roberts⁎ Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853, USA Received 30 November 2010; received in revised form 31 January 2011; accepted 3 February 2011 Available online 18 February 2011 Edited by M. Gottesman Keywords: transcription; sigma-dependent pausing; backtracking; gene regulation
After promoter escape in Escherichia coli, the initiating σ70 factor is retained by core RNA polymerase (RNAP) for at least tens of nucleotides. While it is bound, σ70 can engage a repeat of a promoter DNA element located downstream of the promoter and thereby induce a transcription pause. The σ70-dependent promoter-proximal pause that occurs at all lambdoid phage late gene promoters is essential to regulation of the late gene operons. Several, and possibly many, E. coli promoters have associated σ70 dependent pauses. Clearly characterized σ70 -dependent pauses occur within 25 nucleotides of the start site, but σ70-dependent pausing might occur farther downstream as well. In this review, we summarize evidence for σ70-dependent promoter-proximal and promoter-distal pausing, and we discuss its potential regulatory function and mechanistic basis.
Introduction Initial binding, promoter opening and promoter escape are all known regulatory targets in the initiation of bacterial RNA polymerase (RNAP) but there is a further potential regulatory step before the transition to elongation. After transcription initiation and promoter escape in Escherichia coli, the primary σ factor, σ70, remains associated with core RNAP for some distance and can induce a σ70dependent pause within ~ 25 nt of the start site, and possibly further.1 Pausing occurs at promoters that encode a reiteration of either a promoter – 10 or a promoter – 35 element along with an appropriately spaced class II, or backtrack-inducing, pause sequence.2,3 At late gene promoters of bacteriophage λ and its relatives, this σ 70 -dependent promoter-proximal paused RNAP is modified by phage-encoded Q proteins that convert the enzyme from a terminator-sensitive to a terminator-resistant *Corresponding author. E-mail address: [email protected]. Abbreviations used: RNAP, RNA polymerase; FRET, Förster resonance energy transfer; ChIP, chromatin immunoprecipitation.
© 2011 Elsevier Ltd. All rights reserved.
form; therefore, the pause is essential to the regulation of phage late gene expression. Several bacterial promoters encode such σ 70 -dependent pauses but the σ70-dependent pause has a known regulatory role only at the phage promoters.1,4–6 A large class of eukaryotic promoters encodes promoter-proximally paused or “stalled” complexes within 30–50 nt of the transcription start site.7–9 Stalling at eukaryotic promoters is clearly a mechanistically distinct process but the prevalence of promoter-proximal paused or stalled RNAP complexes in such diverse organisms suggests that they might represent an important regulatory target in gene expression. In this review, we summarize studies that have identified the role of σ70 in pausing and led to the characterization of paused complexes.
σ 70 region 2 induces a promoter-proximal pause at lambdoid phage late gene promoters In transcription from lambdoid phage late gene promoters, modification of RNAP by the late gene antiterminator protein Q occurs at a promoter-
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Review: σ70- dependent Transcription Pausing
proximal paused complex located within 25 nt of the + 1 transcription start site; the pause is at + 16/+ 17 for phage λ, + 25 for phage 82 and at + 18 for phage 21.10–13 Q function requires both its DNA binding element, located between the promoter –10 and – 35 elements, and a paused RNAP complex upon which it can act.14 An important hallmark of Q modification is that the pause decays more quickly in the presence of Q protein than in its absence, implying that Q modification antagonizes the elongation barrier at the pause.11 The function and part of the sequence basis of the pause at lambdoid late gene promoters was understood before the nature of pause formation was known. Several studies utilizing deletion constructs of λpR′ and 82pR′ revealed that the initial transcribed sequence up to the site of pausing is required for pause formation.11,15,16 Analysis of mutants in the early transcribed region showed that positions + 2 and + 6 of λpR′ are responsible for pause formation and are required for Q modification.14,17 λQ can bind either A + 2G or T + 6G DNA as well as wild type, but elongation complexes stalled at the pause site via nucleotide deprivation are modified by λQ only if the template is wild type at these positions.14 Together, these data indicate that simply pausing at +16/+ 17 does not create a substrate for λQ; rather, positions + 2 and +6 are required for the formation of a transcription complex that is susceptible to modification by Q protein. A study that used heteroduplex templates showed that sequences required for pause formation act predominantly in the nontemplate strand.18 RNAP complexes formed on templates with a wild type
Fig. 1. Promoters encoding σ70 region 2-dependent pauses contain – 10-like repeats and class II pause sequences. Left: Promoters that encode σ70 region 2dependent pauses, including the lambdoid phage late gene promoters and several E. coli promoters; the pause position relative to the + 1 transcription start site is noted in parentheses. Right: Promoter sequences are aligned with the – 10-like element (AnnnT); the first uppercase nucleotide shown in each sequence is at position + 1. The backtrack-inducing class II pause sequence up to the pause nucleotide(s) is shown with G/C residues in blue and A/T residues in red. Black bars indicate experimentally determined pause positions; gray bars indicate approximate or estimated pause positions.
783 template strand but a + 6G mutation in the nontemplate strand do not pause at + 16; however, complexes formed on wild type nontemplate strands but with a + 6C mutation in the template strand pause nearly as well as complexes formed on fully wild type templates. The same strand specificity for pause formation is seen for positions + 2, + 5, +7, + 8 and + 9, although there is a substantial effect of the template strand as well for certain sites.18 The requirement for σ70 in pause formation was not established until it was noted that the early transcribed sequence of λpR′ (AACGAT from +1 to +6) resembles a promoter – 10 element (consensus TATAAT; Fig. 1). Matches to two of the most conserved bases of the –10 element, A at the second position and T at the sixth position, occur at +2 and +6, the two bases found to be essential to +16/+17 pause formation and λQ activity.2 Furthermore, the other sequenced lambdoid phage late gene promoters also contain a promoter-proximal match at least to the nAnnnT of the promoter –10 element; phage 82pR′ contains a match to a consensus extended –10 element (TnTGnTATAAT).19 This repeat element is now referred to as the –10-like sequence (Fig. 1). Two convincing experiments confirmed the presence of σ70 at promoter-proximal pauses. First, λpR′ heteroduplex bubble templates were used to test RNAP synthesis through the pause in the presence or in the absence of σ70; these bubble templates support initiation without a promoter and thus without requiring σ70.2 A pause is observed when synthesis on bubble templates occurs with holoenzyme; however, there is no pause if only core RNAP is supplied. As expected, pausing on + 2G or + 6G templates was abolished even in the presence of σ70. Furthermore, the use of σ70 fragments on the bubble templates narrowed the σ70 requirement for pausing to the region 2 residues 360–448, which is known to be the promoter – 10 recognition region. To confirm this result, stalled λ +16/+ 17 or 82 + 25 complexes were formed on homoduplex templates and initiated from the promoter with wild type σ70. Washing these complexes and analyzing them for protein content revealed that σ70 is present in complexes formed on wild type templates but is retained only very weakly on λ + 6G or 82 + 10C mutant templates. Position + 10 is the 82pR′ equivalent of λpR′ + 2 in terms of the –10-like sequence (Fig. 1).2 A screen for residues in σ70 that, when mutated, reduce the antitermination activity of λQ identified mainly residues in region 2.2, confirming the role of region 2 in σ 70 -dependent pausing at phage promoters.20 When modeled on a bacterial RNAP crystal structure, these mutated residues appear to be involved in σ70 region 2 – RNAP core interactions and not in DNA recognition. In addition to a pausing defect, two mutant σ70s (L402F and N409D) were found to weaken the stability of the open complex and greatly reduce abortive initiation; thus, these
784 RNAP mutants are likely to decrease complex stability rather than reducing the affinity of σ70 region 2 for promoter –10 DNA or – 10-like DNA. Another characteristic of σ70-dependent pauses is that overall pause occupancy and pause half-life are reduced in the presence of the transcript cleavage factors GreA and GreB, which rescue backtracked elongation complexes. Thus, at least a component of σ 70 -dependent paused complexes is in a state related to elongation backtracking.21 Importantly, not all σ70-dependent paused complexes are sensitive to Gre factors; whereas the λpR′ 17 nt RNA is nearly eliminated in the presence of GreB in vitro, the 16 nt RNA is insensitive. Furthermore, it is this latter pause that is preferentially chased by λQ. GreA, which is not required for phage growth, was found to enhance Q activity in vivo. It might appear to be paradoxical that Q activity, which requires a paused substrate, is enhanced in the presence of Gre factors that reduce overall pausing; however, this finding suggests that one subset of paused complexes is in a transcriptionally inactive state in which the 3′ end of the RNA is not in the active site and a second subset of paused complexes is transcriptionally competent. The role of GreA and GreB, then, might be to rescue the inactive complexes to regenerate the active ones, thus providing a greater concentration of the substrate for Q modification. These considerations are crucial to the model of σ70-dependent promoterproximal pause formation and are discussed in more detail below.
σ 70 region 2-dependent pausing at E. coli promoters The fact that no phage-specific protein is required for σ70-dependent promoter-proximal pausing suggests that such pausing might occur also at E. coli promoters. Two studies showed that the lac promoter encodes a pause like those observed at lambdoid late gene promoters.4,5 The lac promoter forms a 17 nt or 18 nt paused RNA in vitro; pause formation is dependent on the AnnnT motif located at positions + 2 to + 6 (Fig. 1) and pausing is reduced when the RNAP holoenzyme contains σ70 region 2 mutant L402F or when GreB is present in the reaction, all characteristics of the phage late gene promoter pauses.4,5 Fusing the initial transcribed region of lac to the promoter of λpR′ (including the λQ DNA binding element) results in a pause at + 17/+ 18 in vitro and produces an active substrate for λQ modification in vivo.5 Förster resonance energy transfer (FRET) analysis, in which the efficiency of energy transfer between two fluorophores detects the concentration and proximity of interacting species, determined that σ70 is present in a larger number of complexes formed on wild-type lacUV5 templates than on those containing an A to G
Review: σ70- dependent Transcription Pausing
mutation at + 2 that disrupts the required AnnnT motif. Furthermore, σ70 crosslinks to DNA in a lac promoter-proximal paused complex, indicating that σ70 is present at the pause; the crosslink is abolished on a + 6 mutant template that does not support pausing.4 The lac pause revealed the involvement of a second σ70 domain in region 2-dependent pausing. When it was characterized for complexes formed on scaffolds (in order to make transcribing complexes in the absence of a promoter) in the presence of a series of σ70 fragments, the lac pause was seen only if a σ70 region 1.2/2 fragment was used (residues 94–448) and not if σ70 region 2 alone was used (residues 102–448).22 σ70 region 1.2 binds to the nontemplate strand of the promoter “discriminator” element located just downstream of the promoter – 10 element, contributing to promoter strength.23 Its function is best characterized at rrn promoters, in which the strongest interaction of σ70 region 1.2 with the discriminator element occurs when the second nucleotide just downstream of the promoter – 10 element is guanosine and the weakest interaction occurs when that nucleotide is cytosine. Presumably, this specificity holds true for all promoters, although the binding sequence is only partially characterized. The lambdoid σ70 region 2-dependent pauses are also dependent on σ70 region 1.23 (J. J. Filter and J.W. R., unpublished results), despite an earlier observation that σ70 region 2 alone induces pausing on bubble templates.2 Pause formation is dependent on σ70 region 1.2 interacting with a “discriminator-like” sequence in the nontemplate strand.18 The discriminator-like sequences just downstream of the σ70 region 2-dependent promoter-proximal pause-inducing – 10-like sequences do not all contain the optimal guanosine residue 2 nt downstream from the – 10-like sequence (Fig. 1). However, pausing on λpR′ heteroduplex templates was reduced if each of the nontemplate strand bases in the discriminatorlike GGG sequence (positions + 7 to + 9) was mutated to A, suggesting that, at least for λpR′, σ70 region 1.2 might require a sequence-specific interaction.18 It is not known whether σ70 region 1.2 can induce a promoter-proximal pause in the absence of a σ70 region 2-engaging –10-like sequence. Several additional E. coli promoters have been shown to have σ70 region 2-dependent pauses. Detection of transcription bubbles through the reaction of unpaired thymidines in the unwound DNA with KMnO4 revealed that 7 of 34 promoters showed downstream unpaired DNA, consistent with promoter-proximal pausing.6 These promoters encode an AnnnT motif downstream of the promoter – 10 element and the associated pauses are less efficient in an rpoD-L402F strain and more efficient in a ΔgreA strain. Of the five promoters active in vitro, three of the promoter pauses are reduced if either the
Review: σ70- dependent Transcription Pausing
A or T in the AnnnT sequence is mutated, confirming dependence on the σ70 region 2; KMnO4 reactivity of the other two promoters, for which the –10-like sequence is located within a few nucleotides of the promoter – 10 element, more likely reflects stable initial transcribing complexes and not paused transcripts owing to a displaced – 10-like sequence.24 Because the 34 active promoters were selected at random and three were found to encode a σ70 region 2-dependent promoter-proximal pause, it was suggested that ~ 10% of E. coli promoters encode such pauses.6 A question that remains, then, is if and to what extent these pauses regulate gene expression. Only the tnaA promoter, in addition to λpR′, produces more transcript in an rpoD-L402F strain than wild type, indicating that the presence of a σ70dependent pause does not significantly affect levels of gene expression at most promoters. The regulatory role, if any, of σ70-dependent promoter-proximal pausing is not known at any promoters except for the lambdoid phage late gene promoters.
σ 70 region 4 also induces promoter-proximal pauses With the main requirement of a promoterproximal pause being an interaction between σ70 region 2 and a – 10-like element repeat, it was not surprising when the other primary DNA-binding region of σ70, region 4, was found to interact with a repeat of its promoter element to induce a pause.3 In addition to the σ70 region 2-dependent + 25 pause that occurs at phage 82pR′, a pause exists at + 14/+ 15.12 This + 14/+ 15 paused complex was found to be σ70 region 4-dependent, and it shares many pausing characteristics with the σ70 region 2-dependent pauses.3 First, the + 14/+ 15 pause is dependent on a repeat of the promoter –35 element, a “–35-like sequence” located just downstream of the promoter – 35. Mutation of this – 35-like repeat greatly reduces + 14/+ 15 pause efficiency, whereas increasing its match to a consensus – 35 element increases pause efficiency. Like the effect of the σ70-core destabilizing σ70 L402F mutant on region 2-dependent pauses, the σ70 region 4 mutation L607P that weakens σ70 region 4-RNAP core interactions25 reduces +14/+15 pause efficiency. Lastly, +14/+15 paused complexes appear to exist in two conformations, similar to λpR′ paused complexes, with +15 being sensitive to GreB-mediated cleavage and +14 being relatively insensitive to cleavage. There is much remaining to be understood about the role of σ70 region 4 in pausing. Like the E. coli promoters that were found to contain σ70 region 2-dependent pauses, the region 4-dependent pause at 82pR′ has no known regulatory role. Furthermore, no other promoter has been reported to contain region 4-dependent pauses. However, the 82pR′
785 + 14/+ 15 pause occurs in wild type cells in vivo, indicating that σ 70 region 4-dependent pausing represents a potential variant of σ-dependent regulation at E. coli promoters.3 Unlike the well characterized AnnnT sequence of σ70 region 2-dependent pauses, no subset of essential nucleotides has been identified in the – 35-like consensus. Additionally, it is expected, but not confirmed, that a σ70 region 4-dependent pause should not occur past template position + 16 or + 17; at that length, the growing RNA chain should have displaced the σ70 region 4 from core RNAP,25 possibly preventing it from interacting properly with –35-like repeats.
DNA scrunching in σ 70 -dependent pausing A revealing but seemingly paradoxical fact is that the length of RNA that has been synthesized at the elongation step when the pause-inducing sequence can first engage σ70 is not the same as the length of RNA in the paused complex. For example, at λpR′, σ70 region 2 should engage the –10-like element after RNAP has synthesized 12 nt of RNA (the second nucleotide of the promoter –10 element is located at position –11 and the second nucleotide of the –10like element is located at position +2; the separation is 12 nt). However, the RNA in a λpR′ paused complex is 16 nt or 17 nt long (Fig. 2). Similarly, the 82pR′ pause results in a 25 nt RNA whereas σ70 region 2 engagement should occur after RNAP has translocated 20 nt, and σ70 region 4 engagement should occur after RNAP has translocated 9 nt but the resultant pause contains a 14/+15 nt RNA. This difference can be accounted for if pause formation involves DNA scrunching, a process in which σ70 contacts with DNA elements are retained while the active center of RNAP translocates downstream, looping out template and nontemplate strand DNA; such scrunching occurs during promoter escape and very likely occurs identically in σ70-dependent promoter-proximal pausing.21,26,27 One difference is that for initial transcribing complexes, relaxation of the scrunch gives rise to abortive synthesis as the incipient RNA is released from the complex; however, the RNA in a promoterproximal paused complex is of a sufficient length that it is not aborted, instead the scrunch is relaxed into a “backtracked-like” state, meaning that RNA is extruded through the secondary channel but RNAP itself did not reverse translocate (as described in more detail below). In this model of σ70-dependent downstream scrunching at λpR′, σ 70 region 2 engages the –10-like repeat when the RNA is 12 nt long, and then the RNAP active center translocates downstream 4 nt or 5 nt and σ70 remains bound to the DNA (Fig. 2). The upstream edge of RNAP is positioned where it would be if RNAP had
786
Review: σ70- dependent Transcription Pausing
Fig. 2. σ70-dependent pausing occurs through DNA scrunching. During transcription from the λ late gene promoter, σ70 region 2 interacts with a repeat of the – 10 element in the nontemplate strand (boxed) that is located 12 nt downstream of the promoter – 10 element; the RNA is therefore 12 nt long when σ70 region 2 engages this DNA element. While σ70 region 2 retains contacts with – 10-like DNA, the active center of RNAP translocates downstream 4 nt as template and nontemplate DNA are scrunched out of the enzyme, forming the σ70-dependent paused, scrunched +16 complex with the 3′OH group of the RNA in the active center. Synthesis to +17 can result in either pause escape (not shown) or the destablization of the scrunched complex; relaxation of the scrunch does not disrupt σ70 region 2/–10-like DNA contacts but does displace the 3′ end of the RNA out of the secondary channel in a backtracking-like reaction. σ70 region 4dependent pausing occurs analogously, but with σ70 region 4 interacting with – 35-like repeat DNA. σ70, red; RNAP core, gray; RNA, orange (to + 12) or blue (indicating bases added during scrunching); black bars, promoter – 35 and – 10 elements; open oval, active site. Broken gray guidelines are shown to orient RNAP.
transcribed 12 nt of RNA, but the forward edge is positioned where it is expected to be if 16 nt had been transcribed; ~ 4 nt of template and nontemplate strand DNA are extruded from the enzyme and the transcription bubble increases in size by 4 nt. Probing with KMnO4 to identify transcription bubbles of paused complexes on late gene promoters in vitro and in vivo indicates that the transcription bubble at the pause is larger than that at the open complex.28
As noted above, λ + 16 paused complexes are resistant to GreA and GreB cleavage, suggesting that the 3′ end of the RNA is in the active site, as would be expected in a paused, scrunched complex; λ + 17 complexes are highly sensitive to GreB cleavage, indicating that RNA 3′OH is extruded through the RNAP secondary channel.21 We propose that a λ + 17 complex, although GreB sensitive, is not backtracked in the usual sense (i.e. there was no reverse translocation of the enzyme because σ70 maintains
Review: σ70- dependent Transcription Pausing
the same DNA contacts throughout the process). Instead, if the scrunch relaxes during synthesis from + 16 to + 17, σ70 retains the same DNA contacts, the RNAP active site shifts to the position at which it was located before scrunching (in the λpR′ case this is + 12) and the 3′ end of the RNA is shifted to the secondary channel where it is sensitive to cleavage (Fig. 2). At both the λ and 82 late gene promoters, if RNA synthesis is halted at the pause position via nucleotide deprivation in vitro, GreB cleavage in the absence of NTPs shows that RNAP resides stably at an upstream site equivalent to where σ70 region 2 engages the – 10-like element (e.g. λpR′ + 12), supporting the idea that scrunch relaxation results in a backtracked-like complex.21
A backtrack-inducing sequence is required for σ 70 -dependent promoter-proximal pausing As noted above, the pause-inducing AnnnT motif is present at all promoters identified to have region 2dependent pauses, and a mutation in the A or T residue of the motif greatly reduces pause efficiency. Even if the –10-like element (along with some downstream sequence, as becomes important below) was displaced from its natural location by upstream nucleotide insertions, a pause was observed, albeit often weakly, at the distance expected based on the size of the insertion.2 When the λpR′ early transcribed region was positioned as promoterdistal as 462 nt, a high concentration of σ70 enabled pause formation at the expected location at +462.29 Together, these observations suggest that only σ70 and a –10-like element are required for σ70-dependent pause formation regardless of template location. However, analysis of the σ70 region 4-dependent pause gave a refined view: a distinct element, separate from the –35-like repeat, is required for efficient pausing and, in fact, the same element positions the pause. When the location of the –35-like element was shifted within the promoter region without changing the transcribed sequence, the remaining pause did not shift from +14/+15.3 This result appears to be in conflict with the σ70 region 2 studies mentioned above, in which the –10-like sequence was displaced; however, in those experiments, both the σ70 binding element and an adjacent downstream sequence were moved. In fact, the σ70 region 2-dependent pauses also require this second element. A common characteristic of all known σ70 dependent promoter-proximal pauses is that DNA downstream of the σ 70 binding element sequence contains a G + C-rich sequence followed by an A + T-rich segment, a configuration typical of the backtrack-inducing sequence for class II pauses;30–33 significantly, this class II pause sequence coincides with the position of the σ70-dependent
787 pause (Fig. 1). In the σ70 region 2-dependent pauses, the G + C-rich sequence is also the discriminator-like sequence and is located just 3′ of the – 10-like element. When the backtrack-inducing sequences associated with both the 82pR′ + 14/+ 15 and + 25 pauses are mutated, such that the A + T-rich residues closest to and including the pause site(s) are replaced with hybrid-stabilizing G/C residues, both pauses are reduced significantly, even in the presence of consensus promoter element repeats.3 The reduction in pause efficiency observed for these backtrackinducing sequence mutants is roughly equivalent to the amount of pause reduction observed when the promoter element repeat is mutated, indicating that both the promoter element repeat and the backtrackinducing sequence are required for full pause induction for both σ70 region 2- and 4-dependent pauses. Furthermore, these backtrack-inducing sequences must determine the position of the pause. The requirement of the backtrack-inducing sequence for σ70-dependent pausing has been established but its exact role and the nature of the G + Crich and A + T-rich sequence requirements remain in question. This sequence induces backtracking during unscrunched synthesis, but we propose it has a distinct role during scrunched synthesis; i.e. the sequence (or some component of it) determines the position of the pause in the fully forward, scrunched state. One conjecture is that stabilization of the paused, scrunched state results from a combination of the G + C-rich segment making unwinding of the upstream boundary of the RNA:DNA hybrid difficult, 34 and the terminal rA + rU-rich RNA sequence destabilizing the growing end in the active center.35 As an example of the influence of these sequences on the pause site, both λpR′ and lac pauses are induced by – 10-like elements located at transcript positions + 1→+ 6, but the associated pauses occur 1 nt apart (+ 16/+ 17 versus + 17/+ 18). It could be relevant that sequences near the pause position are rather different for λpR′ and lac: the G + C-rich region of λpR′ is only 3 nt (GGG), the last of which is at + 9; the G + C-rich region of lac is 4 nt (GCGG), the last of which is at + 12. Additionally, lac has an A + Trich sequence downstream of its pause site whereas λpR′ has a G + C-rich sequence (Fig. 1). Further experiments are required to determine how the length and positioning of these G + C-rich and A + T-rich sequences within the backtrack-inducing sequence affect σ70-dependent pausing.
A model for σ 70 -dependent promoter-proximal pausing The identification of the requirement for a backtrack-inducing sequence in σ 70 -dependent promoter-proximal pausing led to refinement of the model for such pausing. We propose that the primary
788 and essential role of the backtrack-inducing sequence in all σ70-dependent pausing is to define the extent to which complexes can be scrunched (Fig. 3; 2→4). The pause occurs (or the extent of scrunching stops) at a template position where the stability of the RNA: DNA hybrid is at a local minimum. The backtrack-inducing sequence could contribute to establishing the pause in a distinct (but not exclusive) way from determining the site of the pause; it is possible this sequence is required to induce RNAP to backtrack before the scrunched paused complex can form. The earlier understanding of σ70-dependent pausing posited that σ70 engagement occurs as RNAP first encounters the σ70 binding sequence, after which synthesis occurs to the pause position with DNA scrunching (Fig. 3; 1→2→4). It is possible, however, that this engagement does not occur during the initial encounter, possibly because synthesis occurs too quickly for σ70 to engage DNA. Instead, a second role of the backtrack-inducing sequence might be to act as a class II pause sequence and induce RNAP to stall and backtrack to a position where σ70 region 2 (or region 4) is now positioned to engage its promoter element repeat, albeit in a transcriptionally inactive complex in which the 3′ end of the RNA is not in the active site (2→3→5). Either the intrinsic cleavage activity of RNAP or Gre-factor-mediated cleavage could rescue
Review: σ70- dependent Transcription Pausing
the arrested complex (5→2) and allow synthesis with scrunching to the pause site (2→4). In fact, these two pathways (σ70 engagement first; backtracking first then σ70 engagement) are not mutually exclusive and both would result in a σ70-dependent paused, scrunched complex; whichever pathway leads to the formation of a scrunched pause at lambdoid late gene promoters, this complex is competent for Q protein modification (4→6). Relaxation of the promoter-proximal scrunch, regardless of how it forms, yields a backtracked-like complex (4→5) that must be rescued via RNA cleavage (5→2) before the paused, scrunched complex can be regenerated (2→4); the complex is physically indistinguishable from a backtracked complex (i.e. 3→5) but it has formed in the absence of RNAP translocation backwards along the DNA template. Further investigations into the order of events in σ70-dependent pausing and the role of the backtrack-inducing sequence are required to determine if only one or both pathways are utilized in inducing a pause.
σ70 retention in elongation complexes and promoter-distal pausing The ability of σ70 to remain associated with elongation complexes at promoter-proximal sites,
Fig. 3. A model for σ70-dependent promoter-proximal pausing. Two pathways could lead to the construction of a σ70 region 2-dependent paused, scrunched complex. In one (2→3→5→2→4), the backtrack-inducing class II pause is encountered because the σ70 region 2 does not engage the –10-like repeat during synthesis. RNAP backtracks to align σ70 region 2 with the –10-like element; the complex then is rescued via intrinsic or Gre factor-mediated RNA cleavage and the DNA-bound RNAP scrunches with synthesis to the end of the class II pause sequence. In a second, nonexclusive, pathway (2→4), σ70 region 2 engages the –10-like repeat during synthesis; the DNA-bound RNAP scrunches with synthesis to the end of the class II pause sequence. Either pathway results in the formation of the substrate for lambdoid phage Q proteins (4→6). Relaxation of the scrunched complex (4→5) yields a backtracked-like complex, in which the 3′ end of the RNA is extruded from the secondary channel but RNAP has not reverse translocated because σ70 – DNA contacts have been maintained. The scrunched complex can be regenerated when the backtracked-like complex is rescued by RNA cleavage, as above (5→2→4). Similar steps occur in σ70 region 4-dependent pausing, but with σ70 region 4 interacting with a –35-like repeat.
Review: σ70- dependent Transcription Pausing
as opposed to an obligatory release during the transition from initiation to elongation, is no longer in question36 but the distance at which σ70 can remain a part of elongation complexes and its effect on downstream elongation are unclear. Numerous structural and biochemical studies suggest that as RNA synthesis proceeds, σ70/RNAP contacts are disrupted, which should weaken the affinity of σ70 for core RNAP: namely, the σ70 region 3 linker that occupies the RNA exit channel should be displaced after synthesis of a few nucleotides and σ70 region 4 should be displaced after the RNA chain has lengthened to about 17 nt.25,37,38 Once the RNA is longer than 17 nt, it is expected that only σ70 regions 1.2 and 2 remain in contact with the core, although σ70 then competes with elongation factors such as NusG and NusA for these binding sites.39,40,41 Studies of early elongating RNAP complexes in vitro indicate that σ70 always remains associated with RNAP core at least as far as +16/+ 17 (likely to + 25). In one study, transcription was initiated on λpR′ with RNAP containing wild type σ70, σ70 L402F or σS, walked to + 16 via nucleotide deprivation, and then washed and restarted in the presence of a large molar excess of either the σ70 used for initiation or a competing σ70 with distinguishable pausing properties. In all cases, the pausing characteristics of the complexes were the same as those observed with the initiating σ70 without an exogenous σ70 challenge, suggesting that the same σ70 molecule directs both initiation and pausing.42 This experiment was not done with an 82pR′ template, the lambdoid phage late gene promoter for which pausing occurs at the greatest distance from + 1, although it is plausible that a similar result would be seen. FRET experiments on lacUV5 with a mutated – 10-like element (to eliminate the contribution of a σ70-retaining sequence) confirmed that up to 90% of complexes contain σ70 at least as far as + 15, and it is presumably the initiating σ70 that is present.5,43 These experiments further showed that 50–60% of complexes at + 50 contain σ70, but the question of retention versus rebinding was not addressed.43 Relative to the late gene promoters of lambdoid phages λ, 80 and 21, the 82pR′ pause occurs 7~9 nt further downstream. It appears to require a consensus extended –10 sequence (TnTGnTAnnnT) for full pause induction; if the underlined T in the extended – 10 sequence is mutated to G or C, + 25 pause efficiency is reduced ~ twofold (S.A.P. and J.W.R., unpublished results). In contrast, other late gene promoter pauses require only the AnnnT motif, suggesting that the further the –10-like sequence is from the + 1 start site the stronger a σ70-engaging sequence is required for paused complex formation. Consistently, if a series of spacers with lengths in the range from 2 to 20 nt is introduced to displace the λ + 16/+ 17 pause from + 19 up to + 37, only a weak pause remains unless an extended – 10 sequence is
789 present.2 When the local concentration of σ70 is increased via a translational fusion of σ70 to the β′ core subunit and is used to transcribe the λ template with a 20 nt spacer, a pause is observed at +37.29 Tethering σ70 to the core was shown to induce a σ70 region 2-dependent pause as far downstream as +462; additional experiments showed that a large molar excess of σ70 can also induce a pause at +462, suggesting that σ70 is no longer retained by the core at those distances from the +1 start site but it can reassociate with the core and display standard σ70 pause characteristics if present at a sufficiently high concentration.29 Several in vivo chromatin immunoprecipitation (ChIP) studies have been used to monitor promoterdistal σ70 retention throughout the genome. In these studies, proteins were chemically crosslinked to DNA and RNAP (the β or β′ subunit) or σ70 was precipitated with specific antibodies; the immunoprecipitated DNA was analyzed via PCR or microarray to identify the regions of the chromosome where these proteins are present. A study of ribosomal operons concluded that σ 70 travels downstream with RNAP and is released from the core stochastically;44 another ChIP-on-chip study of 109 transcription units found σ70 to be retained in the coding regions of nearly all promoters. 41 However, in other studies, retention was seen at only some promoters and only if the cells were grown in rich medium or were treated in the stationary phase.45,46 It is difficult to compare the results of these studies directly because the data were obtained from cells treated under differing growth conditions (mid-log versus stationary phase growth; rich versus minimal medium). Thus, the exact extent of in vivo downstream retention of σ70 is unclear, although these studies are consistent with some retention occurring, depending on the operon and growth conditions. Another caveat to using ChIP to assess σ70 retention is that σ70 makes basespecific contacts with promoter or pause DNA but it is not likely to make these contacts during elongation; thus, crosslinking efficiency might be greater at a promoter or sequence-encoded pause than downstream. Still, these experiments are beginning to shed light on σ70 retention during elongation and might reflect the effects of cellular factors that are absent from in vitro studies. An interesting hypothesis regarding σ70 retention is that one role of σ70-dependent promoter-proximal pausing is to retain σ70 in elongating complexes.5 A – 10-like element was introduced 20 nt downstream of the +16/+ 17 pause-inducing –10-like element in λpR′, and an appropriately displaced ~+ 37 pause was observed in vitro; however, the + 37 pause was much weaker if the upstream – 10-like element was mutated.47 This suggests that the +16/+ 17 pause can function to retain σ70 in the complex, essentially increasing its effective concentration downstream.
790 In fact, pause efficiency is increased at the downstream site to the same extent if an upstream – 10like element is present or if a ~ 30-fold increase in σ70 concentration is used. This study additionally showed via ChIP-PCR that σ70 retention is increased approximately twofold in vivo as far as ~ 700 nt downsteam, but again only if a promoter-proximal σ70-engaging –10-like element is present. σ70 retention was suggested in another study,6 in which the cspA and tnaA operons were shown to have several pauses in the first ~ 100 nt that are reduced in the presence of σ70 L402F, which strongly reduces σ70 region 2-dependent pausing. There are several candidate AnnnT sequences throughout the first ~ 100 nt that could function to retain σ70 for the next; however, mutational analysis of these – 10-like elements and their role in downstream pausing has not been conducted. In contrast, other studies of σ70 in elongation complexes suggest that promoter-proximal retention has no effect on promoter-distal retention. First, in the ChIP experiment noted above, there is no apparent downstream σ70 signal on the lacZYA operon under any growth conditions, despite the promoter-proximal pause identified later at this promoter.46 A single molecule FRET study of the lacUV5 promoter and its pause-deficient + 2G variant indicated that the wild type template initially retains more σ70 in complexes with less than 14 nt of RNA synthesized than do mutant templates but there is no difference between the templates in initial retention of σ70 in complexes with 50 nt of RNA, suggesting that upstream retention has no effect on downstream retention.43 Lastly, the efficiency of pausing for the 82pR′ σ70 region 4-dependent pause at + 14/+ 15 has no effect on the efficiency of pausing at the downstream + 25 region 2-dependent pause, although the former could plausibly have no affect on σ70 retention because pausing is induced by σ70 region 4 rather than region 2.3
Concluding remarks It is clear that σ70-dependent promoter-proximal pausing occurs during transcription from numerous promoters in E. coli. More distal σ70-dependent pausing can occur under special conditions in vitro, such as a higher local concentration of σ70, but it has not been shown clearly to occur in vivo. σ 70 dependent pausing requires only two sequence determinants: a repeat of either the promoter – 10 or the promoter – 35 element, and an appropriately spaced backtrack-inducing (class II pause) sequence; these minimal requirements suggest that pausing could occur throughout the E. coli genome and in other bacteria. With the exception of the lambdoid phage late gene promoters and the requirement of the pause for Q antitermination activity, the function
Review: σ70- dependent Transcription Pausing
of σ70-dependent pausing is unclear; it could equally well reflect accidental occurrence of pause-inducing sequences or an evolved regulatory function. Intriguingly, the mechanistically distinct eukaryotic RNA polymerase II promoter-proximal stalling appears also to require DNA-binding proteins, a related backtrack-inducing sequence and an RNA cleavage factor;7,9,48 these similarities suggest a generally important function of promoter-proximal pausing in transcription. Further studies are required to catalogue bacterial promoters that utilize σ 70 -dependent pausing and to determine any regulatory role it might have. In addition, characterizing the role of the backtrack-inducing sequence, especially the extent to which the G + C-rich and A + T-rich sequences affect scrunching and pause site determination, will be important in understanding the mechanism of σ70-dependent pausing.
Acknowledgements We thank Jeremy Bird for comments and the National Institutes of Health for support.
References 1. Roberts, J. W., Yarnell, W., Bartlett, E., Guo, J., Marr, M., Ko, D. C. et al. (1998). Antitermination by bacteriophage lambda Q protein. Cold Spring Harbor Symp. Quant. Biol. 63, 319–325. 2. Ring, B. Z., Yarnell, W. S. & Roberts, J. W. (1996). Function of E. coli RNA polymerase sigma factor sigma 70 in promoter-proximal pausing. Cell, 86, 485–493. 3. Perdue, S. A. & Roberts, J. W. (2010). A backtrackinducing sequence is an essential component of Escherichia coli sigma70-dependent promoter-proximal pausing. Mol. Microbiol. 78, 636–650. 4. Brodolin, K., Zenkin, N., Mustaev, A., Mamaeva, D. & Heumann, H. (2004). The sigma 70 subunit of RNA polymerase induces lacUV5 promoter-proximal pausing of transcription. Nat. Struct. Mol. Biol. 11, 551–557. 5. Nickels, B. E., Mukhopadhyay, J., Garrity, S. J., Ebright, R. H. & Hochschild, A. (2004). The sigma 70 subunit of RNA polymerase mediates a promoterproximal pause at the lac promoter. Nat. Struct. Mol. Biol. 11, 544–550. 6. Hatoum, A. & Roberts, J. (2008). Prevalence of RNA polymerase stalling at Escherichia coli promoters after open complex formation. Mol. Microbiol. 68, 17–28. 7. Saunders, A., Core, L. J. & Lis, J. T. (2006). Breaking barriers to transcription elongation. Nat. Rev. Mol. Cell Biol. 7, 557–567. 8. Core, L. J. & Lis, J. T. (2008). Transcription regulation through promoter-proximal pausing of RNA polymerase II. Science, 319, 1791–1792. 9. Nechaev, S., Fargo, D. C., dos Santos, G., Liu, L., Gao, Y. & Adelman, K. (2010). Global analysis of short
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RNAs reveals widespread promoter-proximal stalling and arrest of Pol II in Drosophila. Science, 327, 335–338. Dahlberg, J. E. & Blattner, F. R. (1973). In vitro transcription products of lambda DNA: nucleotide sequences and regulatory sites. In Virus Research, Proceedings of the 1973 ICN-UCLA Symposium on Molecular Biology (Fox, C. R. & Robinson, W. S., eds), pp. 533–544, Academic Press, New York. Grayhack, E. J., Yang, X. J., Lau, L. F. & Roberts, J. W. (1985). Phage lambda gene Q antiterminator recognizes RNA polymerase near the promoter and accelerates it through a pause site. Cell, 42, 259–269. Yang, X. J., Goliger, J. A. & Roberts, J. W. (1989). Specificity and mechanism of antitermination by Q proteins of bacteriophages lambda and 82. J. Mol. Biol. 210, 453–460. Guo, H. C., Kainz, M. & Roberts, J. W. (1991). Characterization of the late-gene regulatory region of phage 21. J. Bacteriol. 173, 1554–1560. Yarnell, W. S. & Roberts, J. W. (1992). The phage lambda gene Q transcription antiterminator binds DNA in the late gene promoter as it modifies RNA polymerase. Cell, 69, 1181–1189. Yang, X. J., Hart, C. M., Grayhack, E. J. & Roberts, J. W. (1987). Transcription antitermination by phage lambda gene Q protein requires a DNA segment spanning the RNA start site. Genes Dev. 1, 217–226. Goliger, J. A. & Roberts, J. W. (1989). Sequences required for antitermination by phage 82 Q protein. J. Mol. Biol. 210, 461–471. Yang, X. J. (1988). Transcription antitermination mediated by lambdoid phage Q proteins. PhD thesis, Cornell University, Ithaca, NY. Ring, B. Z. & Roberts, J. W. (1994). Function of a nontranscribed DNA strand site in transcription elongation. Cell, 78, 317–324. Kumar, A., Malloch, R. A., Fujita, N., Smillie, D. A., Ishihama, A. & Hayward, R. S. (1993). The minus 35recognition region of Escherichia coli sigma 70 is inessential for initiation of transcription at an “extended minus 10” promoter. J. Mol. Biol. 232, 406–418. Ko, D. C., Marr, M. T., Guo, J. & Roberts, J. W. (1998). A surface of Escherichia coli sigma 70 required for promoter function and antitermination by phage lambda Q protein. Genes Dev. 12, 3276–3285. Marr, M. T. & Roberts, J. W. (2000). Function of transcription cleavage factors GreA and GreB at a regulatory pause site. Mol. Cell, 6, 1275–1285. Zenkin, N., Kulbachinskiy, A., Yuzenkova, Y., Mustaev, A., Bass, I., Severinov, K. & Brodolin, K. (2007). Region 1.2 of the RNA polymerase sigma subunit controls recognition of the –10 promoter element. EMBO J. 26, 955–964. Haugen, S. P., Berkmen, M. B., Ross, W., Gaal, T., Ward, C. & Gourse, R. L. (2006). rRNA promoter regulation by nonoptimal binding of sigma region 1.2: an additional recognition element for RNA polymerase. Cell, 125, 1069–1082. Margeat, E., Kapanidis, A. N., Tinnefeld, P., Wang, Y., Mukhopadhyay, J., Ebright, R. H. & Weiss, S. (2006). Direct observation of abortive initiation and promoter escape within single immobilized transcription complexes. Biophys. J. 90, 1419–1431.
791 25. Nickels, B. E., Garrity, S. J., Mekler, V., Minakhin, L., Severinov, K., Ebright, R. H. & Hochschild, A. (2005). The interaction between sigma70 and the beta-flap of Escherichia coli RNA polymerase inhibits extension of nascent RNA during early elongation. Proc. Natl Acad. Sci. USA, 102, 4488–4493. 26. Kapanidis, A. N., Margeat, E., Ho, S. O., Kortkhonjia, E., Weiss, S. & Ebright, R. H. (2006). Initial transcription by RNA polymerase proceeds through a DNAscrunching mechanism. Science, 314, 1144–1147. 27. Revyakin, A., Liu, C., Ebright, R. H. & Strick, T. R. (2006). Abortive initiation and productive initiation by RNA polymerase involve DNA scrunching. Science, 314, 1139–1143. 28. Kainz, M. & Roberts, J. (1992). Structure of transcription elongation complexes in vivo. Science, 255, 838–841. 29. Mooney, R. A. & Landick, R. (2003). Tethering sigma70 to RNA polymerase reveals high in vivo activity of sigma factors and sigma70-dependent pausing at promoter-distal locations. Genes Dev. 17, 2839–2851. 30. Nudler, E., Mustaev, A., Lukhtanov, E. & Goldfarb, A. (1997). The RNA-DNA hybrid maintains the register of transcription by preventing backtracking of RNA polymerase. Cell, 89, 33–41. 31. Guajardo, R. & Sousa, R. (1997). A model for the mechanism of polymerase translocation.J. Mol. Biol. 265, 8; Genes Dev 17, 283919. 32. Komissarova, N. & Kashlev, M. (1997). RNA polymerase switches between inactivated and activated states by translocating back and forth along the DNA and the RNA. J. Biol. Chem. 272, 15329–15338. 33. Artsimovitch, I. & Landick, R. (2000). Pausing by bacterial RNA polymerase is mediated by mechanistically distinct classes of signals. Proc. Natl Acad. Sci. USA, 97, 7090–7095. 34. Gilbert, W., Maizels, N. & Maxam, A. (1974). Sequences of controlling regions of the lactose operon. Cold Spring Harbor Symp. Quant. Biol. 38, 845–855. 35. Toulokhonov, I., Zhang, J., Palangat, M. & Landick, R. (2007). A central role of the RNA polymerase trigger loop in active-site rearrangement during transcriptional pausing. Mol. Cell, 27, 406–419. 36. Mooney, R. A., Darst, S. A. & Landick, R. (2005). Sigma and RNA polymerase: an on-again, off-again relationship? Mol. Cell, 20, 335–345. 37. Murakami, K. S., Masuda, S., Campbell, E. A., Muzzin, O. & Darst, S. A. (2002). Structural basis of transcription initiation: an RNA polymerase holoenzyme-DNA complex. Science, 296, 1285–1290. 38. Vassylyev, D. G., Sekine, S., Laptenko, O., Lee, J., Vassylyeva, M. N., Borukhov, S. & Yokoyama, S. (2002). Crystal structure of a bacterial RNA polymerase holoenzyme at 2.6 Å resolution. Nature, 417, 712–719. 39. Greenblatt, J. & Li, J. (1981). Interaction of the sigma factor and the nusA gene protein of E. coli with RNA polymerase in the initiation-termination cycle of transcription. Cell, 24, 421–428. 40. Belogurov, G. A., Vassylyeva, M. N., Svetlov, V., Klyuyev, S., Grishin, N. V., Vassylyev, D. G. & Artsimovitch, I. (2007). Structural basis for converting
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a general transcription factor into an operon-specific virulence regulator. Mol. Cell, 26, 117–129. Mooney, R. A., Davis, S. E., Peters, J. M., Rowland, J. L., Ansari, A. Z. & Landick, R. (2009). Regulator trafficking on bacterial transcription units in vivo. Mol. Cell, 33, 97–108. Marr, M. T., Datwyler, S. A., Meares, C. F. & Roberts, J. W. (2001). Restructuring of an RNA polymerase holoenzyme elongation complex by lambdoid phage Q proteins. Proc. Natl Acad. Sci. USA, 98, 8972–8978. Kapanidis, A. N., Margeat, E., Laurence, T. A., Doose, S., Ho, S. O., Mukhopadhyay, J. et al. (2005). Retention of transcription initiation factor sigma70 in transcription elongation: single-molecule analysis. Mol. Cell, 20, 347–356. Raffaelle, M., Kanin, E. I., Vogt, J., Burgess, R. R. & Ansari, A. Z. (2005). Holoenzyme switching and
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stochastic release of sigma factors from RNA polymerase in vivo. Mol. Cell, 20, 357–366. Reppas, N. B., Wade, J. T., Church, G. M. & Struhl, K. (2006). The transition between transcriptional initiation and elongation in E. coli is highly variable and often rate limiting. Mol. Cell, 24, 747–757. Wade, J. T. & Struhl, K. (2004). Association of RNA polymerase with transcribed regions in Escherichia coli. Proc. Natl Acad. Sci. USA, 101, 17777–17782. Deighan, P., Pukhrambam, C., Nickels, B. E. & Hochschild, A. (2011). Initial transcribed region sequences influence the composition and functional properties of the bacterial elongation complex. Genes Dev. 25, 77–88. Adelman, K., Marr, M. T., Werner, J., Saunders, A., Ni, Z., Andrulis, E. D. & Lis, J. T. (2005). Efficient release from promoter-proximal stall sites requires transcript cleavage factor TFIIS. Mol. Cell, 17, 103–112.
J. Mol. Biol. (2011) 412, 782–792 Contents lists available at www.sciencedirect.com
Journal of Molecular Biology j o u r n a l h o m e p a g e : h t t p : / / e e s . e l s e v i e r. c o m . j m b
REVIEW
σ 70 -dependent Transcription Pausing in Escherichia coli Sarah A. Perdue and Jeffrey W. Roberts⁎ Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853, USA Received 30 November 2010; received in revised form 31 January 2011; accepted 3 February 2011 Available online 18 February 2011 Edited by M. Gottesman Keywords: transcription; sigma-dependent pausing; backtracking; gene regulation
After promoter escape in Escherichia coli, the initiating σ70 factor is retained by core RNA polymerase (RNAP) for at least tens of nucleotides. While it is bound, σ70 can engage a repeat of a promoter DNA element located downstream of the promoter and thereby induce a transcription pause. The σ70-dependent promoter-proximal pause that occurs at all lambdoid phage late gene promoters is essential to regulation of the late gene operons. Several, and possibly many, E. coli promoters have associated σ70 dependent pauses. Clearly characterized σ70 -dependent pauses occur within 25 nucleotides of the start site, but σ70-dependent pausing might occur farther downstream as well. In this review, we summarize evidence for σ70-dependent promoter-proximal and promoter-distal pausing, and we discuss its potential regulatory function and mechanistic basis.
Introduction Initial binding, promoter opening and promoter escape are all known regulatory targets in the initiation of bacterial RNA polymerase (RNAP) but there is a further potential regulatory step before the transition to elongation. After transcription initiation and promoter escape in Escherichia coli, the primary σ factor, σ70, remains associated with core RNAP for some distance and can induce a σ70dependent pause within ~ 25 nt of the start site, and possibly further.1 Pausing occurs at promoters that encode a reiteration of either a promoter – 10 or a promoter – 35 element along with an appropriately spaced class II, or backtrack-inducing, pause sequence.2,3 At late gene promoters of bacteriophage λ and its relatives, this σ 70 -dependent promoter-proximal paused RNAP is modified by phage-encoded Q proteins that convert the enzyme from a terminator-sensitive to a terminator-resistant *Corresponding author. E-mail address: [email protected]. Abbreviations used: RNAP, RNA polymerase; FRET, Förster resonance energy transfer; ChIP, chromatin immunoprecipitation.
© 2011 Elsevier Ltd. All rights reserved.
form; therefore, the pause is essential to the regulation of phage late gene expression. Several bacterial promoters encode such σ 70 -dependent pauses but the σ70-dependent pause has a known regulatory role only at the phage promoters.1,4–6 A large class of eukaryotic promoters encodes promoter-proximally paused or “stalled” complexes within 30–50 nt of the transcription start site.7–9 Stalling at eukaryotic promoters is clearly a mechanistically distinct process but the prevalence of promoter-proximal paused or stalled RNAP complexes in such diverse organisms suggests that they might represent an important regulatory target in gene expression. In this review, we summarize studies that have identified the role of σ70 in pausing and led to the characterization of paused complexes.
σ 70 region 2 induces a promoter-proximal pause at lambdoid phage late gene promoters In transcription from lambdoid phage late gene promoters, modification of RNAP by the late gene antiterminator protein Q occurs at a promoter-
0022-2836/$ - see front matter © 2011 Elsevier Ltd. All rights reserved.
Review: σ70- dependent Transcription Pausing
proximal paused complex located within 25 nt of the + 1 transcription start site; the pause is at + 16/+ 17 for phage λ, + 25 for phage 82 and at + 18 for phage 21.10–13 Q function requires both its DNA binding element, located between the promoter –10 and – 35 elements, and a paused RNAP complex upon which it can act.14 An important hallmark of Q modification is that the pause decays more quickly in the presence of Q protein than in its absence, implying that Q modification antagonizes the elongation barrier at the pause.11 The function and part of the sequence basis of the pause at lambdoid late gene promoters was understood before the nature of pause formation was known. Several studies utilizing deletion constructs of λpR′ and 82pR′ revealed that the initial transcribed sequence up to the site of pausing is required for pause formation.11,15,16 Analysis of mutants in the early transcribed region showed that positions + 2 and + 6 of λpR′ are responsible for pause formation and are required for Q modification.14,17 λQ can bind either A + 2G or T + 6G DNA as well as wild type, but elongation complexes stalled at the pause site via nucleotide deprivation are modified by λQ only if the template is wild type at these positions.14 Together, these data indicate that simply pausing at +16/+ 17 does not create a substrate for λQ; rather, positions + 2 and +6 are required for the formation of a transcription complex that is susceptible to modification by Q protein. A study that used heteroduplex templates showed that sequences required for pause formation act predominantly in the nontemplate strand.18 RNAP complexes formed on templates with a wild type
Fig. 1. Promoters encoding σ70 region 2-dependent pauses contain – 10-like repeats and class II pause sequences. Left: Promoters that encode σ70 region 2dependent pauses, including the lambdoid phage late gene promoters and several E. coli promoters; the pause position relative to the + 1 transcription start site is noted in parentheses. Right: Promoter sequences are aligned with the – 10-like element (AnnnT); the first uppercase nucleotide shown in each sequence is at position + 1. The backtrack-inducing class II pause sequence up to the pause nucleotide(s) is shown with G/C residues in blue and A/T residues in red. Black bars indicate experimentally determined pause positions; gray bars indicate approximate or estimated pause positions.
783 template strand but a + 6G mutation in the nontemplate strand do not pause at + 16; however, complexes formed on wild type nontemplate strands but with a + 6C mutation in the template strand pause nearly as well as complexes formed on fully wild type templates. The same strand specificity for pause formation is seen for positions + 2, + 5, +7, + 8 and + 9, although there is a substantial effect of the template strand as well for certain sites.18 The requirement for σ70 in pause formation was not established until it was noted that the early transcribed sequence of λpR′ (AACGAT from +1 to +6) resembles a promoter – 10 element (consensus TATAAT; Fig. 1). Matches to two of the most conserved bases of the –10 element, A at the second position and T at the sixth position, occur at +2 and +6, the two bases found to be essential to +16/+17 pause formation and λQ activity.2 Furthermore, the other sequenced lambdoid phage late gene promoters also contain a promoter-proximal match at least to the nAnnnT of the promoter –10 element; phage 82pR′ contains a match to a consensus extended –10 element (TnTGnTATAAT).19 This repeat element is now referred to as the –10-like sequence (Fig. 1). Two convincing experiments confirmed the presence of σ70 at promoter-proximal pauses. First, λpR′ heteroduplex bubble templates were used to test RNAP synthesis through the pause in the presence or in the absence of σ70; these bubble templates support initiation without a promoter and thus without requiring σ70.2 A pause is observed when synthesis on bubble templates occurs with holoenzyme; however, there is no pause if only core RNAP is supplied. As expected, pausing on + 2G or + 6G templates was abolished even in the presence of σ70. Furthermore, the use of σ70 fragments on the bubble templates narrowed the σ70 requirement for pausing to the region 2 residues 360–448, which is known to be the promoter – 10 recognition region. To confirm this result, stalled λ +16/+ 17 or 82 + 25 complexes were formed on homoduplex templates and initiated from the promoter with wild type σ70. Washing these complexes and analyzing them for protein content revealed that σ70 is present in complexes formed on wild type templates but is retained only very weakly on λ + 6G or 82 + 10C mutant templates. Position + 10 is the 82pR′ equivalent of λpR′ + 2 in terms of the –10-like sequence (Fig. 1).2 A screen for residues in σ70 that, when mutated, reduce the antitermination activity of λQ identified mainly residues in region 2.2, confirming the role of region 2 in σ 70 -dependent pausing at phage promoters.20 When modeled on a bacterial RNAP crystal structure, these mutated residues appear to be involved in σ70 region 2 – RNAP core interactions and not in DNA recognition. In addition to a pausing defect, two mutant σ70s (L402F and N409D) were found to weaken the stability of the open complex and greatly reduce abortive initiation; thus, these
784 RNAP mutants are likely to decrease complex stability rather than reducing the affinity of σ70 region 2 for promoter –10 DNA or – 10-like DNA. Another characteristic of σ70-dependent pauses is that overall pause occupancy and pause half-life are reduced in the presence of the transcript cleavage factors GreA and GreB, which rescue backtracked elongation complexes. Thus, at least a component of σ 70 -dependent paused complexes is in a state related to elongation backtracking.21 Importantly, not all σ70-dependent paused complexes are sensitive to Gre factors; whereas the λpR′ 17 nt RNA is nearly eliminated in the presence of GreB in vitro, the 16 nt RNA is insensitive. Furthermore, it is this latter pause that is preferentially chased by λQ. GreA, which is not required for phage growth, was found to enhance Q activity in vivo. It might appear to be paradoxical that Q activity, which requires a paused substrate, is enhanced in the presence of Gre factors that reduce overall pausing; however, this finding suggests that one subset of paused complexes is in a transcriptionally inactive state in which the 3′ end of the RNA is not in the active site and a second subset of paused complexes is transcriptionally competent. The role of GreA and GreB, then, might be to rescue the inactive complexes to regenerate the active ones, thus providing a greater concentration of the substrate for Q modification. These considerations are crucial to the model of σ70-dependent promoterproximal pause formation and are discussed in more detail below.
σ 70 region 2-dependent pausing at E. coli promoters The fact that no phage-specific protein is required for σ70-dependent promoter-proximal pausing suggests that such pausing might occur also at E. coli promoters. Two studies showed that the lac promoter encodes a pause like those observed at lambdoid late gene promoters.4,5 The lac promoter forms a 17 nt or 18 nt paused RNA in vitro; pause formation is dependent on the AnnnT motif located at positions + 2 to + 6 (Fig. 1) and pausing is reduced when the RNAP holoenzyme contains σ70 region 2 mutant L402F or when GreB is present in the reaction, all characteristics of the phage late gene promoter pauses.4,5 Fusing the initial transcribed region of lac to the promoter of λpR′ (including the λQ DNA binding element) results in a pause at + 17/+ 18 in vitro and produces an active substrate for λQ modification in vivo.5 Förster resonance energy transfer (FRET) analysis, in which the efficiency of energy transfer between two fluorophores detects the concentration and proximity of interacting species, determined that σ70 is present in a larger number of complexes formed on wild-type lacUV5 templates than on those containing an A to G
Review: σ70- dependent Transcription Pausing
mutation at + 2 that disrupts the required AnnnT motif. Furthermore, σ70 crosslinks to DNA in a lac promoter-proximal paused complex, indicating that σ70 is present at the pause; the crosslink is abolished on a + 6 mutant template that does not support pausing.4 The lac pause revealed the involvement of a second σ70 domain in region 2-dependent pausing. When it was characterized for complexes formed on scaffolds (in order to make transcribing complexes in the absence of a promoter) in the presence of a series of σ70 fragments, the lac pause was seen only if a σ70 region 1.2/2 fragment was used (residues 94–448) and not if σ70 region 2 alone was used (residues 102–448).22 σ70 region 1.2 binds to the nontemplate strand of the promoter “discriminator” element located just downstream of the promoter – 10 element, contributing to promoter strength.23 Its function is best characterized at rrn promoters, in which the strongest interaction of σ70 region 1.2 with the discriminator element occurs when the second nucleotide just downstream of the promoter – 10 element is guanosine and the weakest interaction occurs when that nucleotide is cytosine. Presumably, this specificity holds true for all promoters, although the binding sequence is only partially characterized. The lambdoid σ70 region 2-dependent pauses are also dependent on σ70 region 1.23 (J. J. Filter and J.W. R., unpublished results), despite an earlier observation that σ70 region 2 alone induces pausing on bubble templates.2 Pause formation is dependent on σ70 region 1.2 interacting with a “discriminator-like” sequence in the nontemplate strand.18 The discriminator-like sequences just downstream of the σ70 region 2-dependent promoter-proximal pause-inducing – 10-like sequences do not all contain the optimal guanosine residue 2 nt downstream from the – 10-like sequence (Fig. 1). However, pausing on λpR′ heteroduplex templates was reduced if each of the nontemplate strand bases in the discriminatorlike GGG sequence (positions + 7 to + 9) was mutated to A, suggesting that, at least for λpR′, σ70 region 1.2 might require a sequence-specific interaction.18 It is not known whether σ70 region 1.2 can induce a promoter-proximal pause in the absence of a σ70 region 2-engaging –10-like sequence. Several additional E. coli promoters have been shown to have σ70 region 2-dependent pauses. Detection of transcription bubbles through the reaction of unpaired thymidines in the unwound DNA with KMnO4 revealed that 7 of 34 promoters showed downstream unpaired DNA, consistent with promoter-proximal pausing.6 These promoters encode an AnnnT motif downstream of the promoter – 10 element and the associated pauses are less efficient in an rpoD-L402F strain and more efficient in a ΔgreA strain. Of the five promoters active in vitro, three of the promoter pauses are reduced if either the
Review: σ70- dependent Transcription Pausing
A or T in the AnnnT sequence is mutated, confirming dependence on the σ70 region 2; KMnO4 reactivity of the other two promoters, for which the –10-like sequence is located within a few nucleotides of the promoter – 10 element, more likely reflects stable initial transcribing complexes and not paused transcripts owing to a displaced – 10-like sequence.24 Because the 34 active promoters were selected at random and three were found to encode a σ70 region 2-dependent promoter-proximal pause, it was suggested that ~ 10% of E. coli promoters encode such pauses.6 A question that remains, then, is if and to what extent these pauses regulate gene expression. Only the tnaA promoter, in addition to λpR′, produces more transcript in an rpoD-L402F strain than wild type, indicating that the presence of a σ70dependent pause does not significantly affect levels of gene expression at most promoters. The regulatory role, if any, of σ70-dependent promoter-proximal pausing is not known at any promoters except for the lambdoid phage late gene promoters.
σ 70 region 4 also induces promoter-proximal pauses With the main requirement of a promoterproximal pause being an interaction between σ70 region 2 and a – 10-like element repeat, it was not surprising when the other primary DNA-binding region of σ70, region 4, was found to interact with a repeat of its promoter element to induce a pause.3 In addition to the σ70 region 2-dependent + 25 pause that occurs at phage 82pR′, a pause exists at + 14/+ 15.12 This + 14/+ 15 paused complex was found to be σ70 region 4-dependent, and it shares many pausing characteristics with the σ70 region 2-dependent pauses.3 First, the + 14/+ 15 pause is dependent on a repeat of the promoter –35 element, a “–35-like sequence” located just downstream of the promoter – 35. Mutation of this – 35-like repeat greatly reduces + 14/+ 15 pause efficiency, whereas increasing its match to a consensus – 35 element increases pause efficiency. Like the effect of the σ70-core destabilizing σ70 L402F mutant on region 2-dependent pauses, the σ70 region 4 mutation L607P that weakens σ70 region 4-RNAP core interactions25 reduces +14/+15 pause efficiency. Lastly, +14/+15 paused complexes appear to exist in two conformations, similar to λpR′ paused complexes, with +15 being sensitive to GreB-mediated cleavage and +14 being relatively insensitive to cleavage. There is much remaining to be understood about the role of σ70 region 4 in pausing. Like the E. coli promoters that were found to contain σ70 region 2-dependent pauses, the region 4-dependent pause at 82pR′ has no known regulatory role. Furthermore, no other promoter has been reported to contain region 4-dependent pauses. However, the 82pR′
785 + 14/+ 15 pause occurs in wild type cells in vivo, indicating that σ 70 region 4-dependent pausing represents a potential variant of σ-dependent regulation at E. coli promoters.3 Unlike the well characterized AnnnT sequence of σ70 region 2-dependent pauses, no subset of essential nucleotides has been identified in the – 35-like consensus. Additionally, it is expected, but not confirmed, that a σ70 region 4-dependent pause should not occur past template position + 16 or + 17; at that length, the growing RNA chain should have displaced the σ70 region 4 from core RNAP,25 possibly preventing it from interacting properly with –35-like repeats.
DNA scrunching in σ 70 -dependent pausing A revealing but seemingly paradoxical fact is that the length of RNA that has been synthesized at the elongation step when the pause-inducing sequence can first engage σ70 is not the same as the length of RNA in the paused complex. For example, at λpR′, σ70 region 2 should engage the –10-like element after RNAP has synthesized 12 nt of RNA (the second nucleotide of the promoter –10 element is located at position –11 and the second nucleotide of the –10like element is located at position +2; the separation is 12 nt). However, the RNA in a λpR′ paused complex is 16 nt or 17 nt long (Fig. 2). Similarly, the 82pR′ pause results in a 25 nt RNA whereas σ70 region 2 engagement should occur after RNAP has translocated 20 nt, and σ70 region 4 engagement should occur after RNAP has translocated 9 nt but the resultant pause contains a 14/+15 nt RNA. This difference can be accounted for if pause formation involves DNA scrunching, a process in which σ70 contacts with DNA elements are retained while the active center of RNAP translocates downstream, looping out template and nontemplate strand DNA; such scrunching occurs during promoter escape and very likely occurs identically in σ70-dependent promoter-proximal pausing.21,26,27 One difference is that for initial transcribing complexes, relaxation of the scrunch gives rise to abortive synthesis as the incipient RNA is released from the complex; however, the RNA in a promoterproximal paused complex is of a sufficient length that it is not aborted, instead the scrunch is relaxed into a “backtracked-like” state, meaning that RNA is extruded through the secondary channel but RNAP itself did not reverse translocate (as described in more detail below). In this model of σ70-dependent downstream scrunching at λpR′, σ 70 region 2 engages the –10-like repeat when the RNA is 12 nt long, and then the RNAP active center translocates downstream 4 nt or 5 nt and σ70 remains bound to the DNA (Fig. 2). The upstream edge of RNAP is positioned where it would be if RNAP had
786
Review: σ70- dependent Transcription Pausing
Fig. 2. σ70-dependent pausing occurs through DNA scrunching. During transcription from the λ late gene promoter, σ70 region 2 interacts with a repeat of the – 10 element in the nontemplate strand (boxed) that is located 12 nt downstream of the promoter – 10 element; the RNA is therefore 12 nt long when σ70 region 2 engages this DNA element. While σ70 region 2 retains contacts with – 10-like DNA, the active center of RNAP translocates downstream 4 nt as template and nontemplate DNA are scrunched out of the enzyme, forming the σ70-dependent paused, scrunched +16 complex with the 3′OH group of the RNA in the active center. Synthesis to +17 can result in either pause escape (not shown) or the destablization of the scrunched complex; relaxation of the scrunch does not disrupt σ70 region 2/–10-like DNA contacts but does displace the 3′ end of the RNA out of the secondary channel in a backtracking-like reaction. σ70 region 4dependent pausing occurs analogously, but with σ70 region 4 interacting with – 35-like repeat DNA. σ70, red; RNAP core, gray; RNA, orange (to + 12) or blue (indicating bases added during scrunching); black bars, promoter – 35 and – 10 elements; open oval, active site. Broken gray guidelines are shown to orient RNAP.
transcribed 12 nt of RNA, but the forward edge is positioned where it is expected to be if 16 nt had been transcribed; ~ 4 nt of template and nontemplate strand DNA are extruded from the enzyme and the transcription bubble increases in size by 4 nt. Probing with KMnO4 to identify transcription bubbles of paused complexes on late gene promoters in vitro and in vivo indicates that the transcription bubble at the pause is larger than that at the open complex.28
As noted above, λ + 16 paused complexes are resistant to GreA and GreB cleavage, suggesting that the 3′ end of the RNA is in the active site, as would be expected in a paused, scrunched complex; λ + 17 complexes are highly sensitive to GreB cleavage, indicating that RNA 3′OH is extruded through the RNAP secondary channel.21 We propose that a λ + 17 complex, although GreB sensitive, is not backtracked in the usual sense (i.e. there was no reverse translocation of the enzyme because σ70 maintains
Review: σ70- dependent Transcription Pausing
the same DNA contacts throughout the process). Instead, if the scrunch relaxes during synthesis from + 16 to + 17, σ70 retains the same DNA contacts, the RNAP active site shifts to the position at which it was located before scrunching (in the λpR′ case this is + 12) and the 3′ end of the RNA is shifted to the secondary channel where it is sensitive to cleavage (Fig. 2). At both the λ and 82 late gene promoters, if RNA synthesis is halted at the pause position via nucleotide deprivation in vitro, GreB cleavage in the absence of NTPs shows that RNAP resides stably at an upstream site equivalent to where σ70 region 2 engages the – 10-like element (e.g. λpR′ + 12), supporting the idea that scrunch relaxation results in a backtracked-like complex.21
A backtrack-inducing sequence is required for σ 70 -dependent promoter-proximal pausing As noted above, the pause-inducing AnnnT motif is present at all promoters identified to have region 2dependent pauses, and a mutation in the A or T residue of the motif greatly reduces pause efficiency. Even if the –10-like element (along with some downstream sequence, as becomes important below) was displaced from its natural location by upstream nucleotide insertions, a pause was observed, albeit often weakly, at the distance expected based on the size of the insertion.2 When the λpR′ early transcribed region was positioned as promoterdistal as 462 nt, a high concentration of σ70 enabled pause formation at the expected location at +462.29 Together, these observations suggest that only σ70 and a –10-like element are required for σ70-dependent pause formation regardless of template location. However, analysis of the σ70 region 4-dependent pause gave a refined view: a distinct element, separate from the –35-like repeat, is required for efficient pausing and, in fact, the same element positions the pause. When the location of the –35-like element was shifted within the promoter region without changing the transcribed sequence, the remaining pause did not shift from +14/+15.3 This result appears to be in conflict with the σ70 region 2 studies mentioned above, in which the –10-like sequence was displaced; however, in those experiments, both the σ70 binding element and an adjacent downstream sequence were moved. In fact, the σ70 region 2-dependent pauses also require this second element. A common characteristic of all known σ70 dependent promoter-proximal pauses is that DNA downstream of the σ 70 binding element sequence contains a G + C-rich sequence followed by an A + T-rich segment, a configuration typical of the backtrack-inducing sequence for class II pauses;30–33 significantly, this class II pause sequence coincides with the position of the σ70-dependent
787 pause (Fig. 1). In the σ70 region 2-dependent pauses, the G + C-rich sequence is also the discriminator-like sequence and is located just 3′ of the – 10-like element. When the backtrack-inducing sequences associated with both the 82pR′ + 14/+ 15 and + 25 pauses are mutated, such that the A + T-rich residues closest to and including the pause site(s) are replaced with hybrid-stabilizing G/C residues, both pauses are reduced significantly, even in the presence of consensus promoter element repeats.3 The reduction in pause efficiency observed for these backtrackinducing sequence mutants is roughly equivalent to the amount of pause reduction observed when the promoter element repeat is mutated, indicating that both the promoter element repeat and the backtrackinducing sequence are required for full pause induction for both σ70 region 2- and 4-dependent pauses. Furthermore, these backtrack-inducing sequences must determine the position of the pause. The requirement of the backtrack-inducing sequence for σ70-dependent pausing has been established but its exact role and the nature of the G + Crich and A + T-rich sequence requirements remain in question. This sequence induces backtracking during unscrunched synthesis, but we propose it has a distinct role during scrunched synthesis; i.e. the sequence (or some component of it) determines the position of the pause in the fully forward, scrunched state. One conjecture is that stabilization of the paused, scrunched state results from a combination of the G + C-rich segment making unwinding of the upstream boundary of the RNA:DNA hybrid difficult, 34 and the terminal rA + rU-rich RNA sequence destabilizing the growing end in the active center.35 As an example of the influence of these sequences on the pause site, both λpR′ and lac pauses are induced by – 10-like elements located at transcript positions + 1→+ 6, but the associated pauses occur 1 nt apart (+ 16/+ 17 versus + 17/+ 18). It could be relevant that sequences near the pause position are rather different for λpR′ and lac: the G + C-rich region of λpR′ is only 3 nt (GGG), the last of which is at + 9; the G + C-rich region of lac is 4 nt (GCGG), the last of which is at + 12. Additionally, lac has an A + Trich sequence downstream of its pause site whereas λpR′ has a G + C-rich sequence (Fig. 1). Further experiments are required to determine how the length and positioning of these G + C-rich and A + T-rich sequences within the backtrack-inducing sequence affect σ70-dependent pausing.
A model for σ 70 -dependent promoter-proximal pausing The identification of the requirement for a backtrack-inducing sequence in σ 70 -dependent promoter-proximal pausing led to refinement of the model for such pausing. We propose that the primary
788 and essential role of the backtrack-inducing sequence in all σ70-dependent pausing is to define the extent to which complexes can be scrunched (Fig. 3; 2→4). The pause occurs (or the extent of scrunching stops) at a template position where the stability of the RNA: DNA hybrid is at a local minimum. The backtrack-inducing sequence could contribute to establishing the pause in a distinct (but not exclusive) way from determining the site of the pause; it is possible this sequence is required to induce RNAP to backtrack before the scrunched paused complex can form. The earlier understanding of σ70-dependent pausing posited that σ70 engagement occurs as RNAP first encounters the σ70 binding sequence, after which synthesis occurs to the pause position with DNA scrunching (Fig. 3; 1→2→4). It is possible, however, that this engagement does not occur during the initial encounter, possibly because synthesis occurs too quickly for σ70 to engage DNA. Instead, a second role of the backtrack-inducing sequence might be to act as a class II pause sequence and induce RNAP to stall and backtrack to a position where σ70 region 2 (or region 4) is now positioned to engage its promoter element repeat, albeit in a transcriptionally inactive complex in which the 3′ end of the RNA is not in the active site (2→3→5). Either the intrinsic cleavage activity of RNAP or Gre-factor-mediated cleavage could rescue
Review: σ70- dependent Transcription Pausing
the arrested complex (5→2) and allow synthesis with scrunching to the pause site (2→4). In fact, these two pathways (σ70 engagement first; backtracking first then σ70 engagement) are not mutually exclusive and both would result in a σ70-dependent paused, scrunched complex; whichever pathway leads to the formation of a scrunched pause at lambdoid late gene promoters, this complex is competent for Q protein modification (4→6). Relaxation of the promoter-proximal scrunch, regardless of how it forms, yields a backtracked-like complex (4→5) that must be rescued via RNA cleavage (5→2) before the paused, scrunched complex can be regenerated (2→4); the complex is physically indistinguishable from a backtracked complex (i.e. 3→5) but it has formed in the absence of RNAP translocation backwards along the DNA template. Further investigations into the order of events in σ70-dependent pausing and the role of the backtrack-inducing sequence are required to determine if only one or both pathways are utilized in inducing a pause.
σ70 retention in elongation complexes and promoter-distal pausing The ability of σ70 to remain associated with elongation complexes at promoter-proximal sites,
Fig. 3. A model for σ70-dependent promoter-proximal pausing. Two pathways could lead to the construction of a σ70 region 2-dependent paused, scrunched complex. In one (2→3→5→2→4), the backtrack-inducing class II pause is encountered because the σ70 region 2 does not engage the –10-like repeat during synthesis. RNAP backtracks to align σ70 region 2 with the –10-like element; the complex then is rescued via intrinsic or Gre factor-mediated RNA cleavage and the DNA-bound RNAP scrunches with synthesis to the end of the class II pause sequence. In a second, nonexclusive, pathway (2→4), σ70 region 2 engages the –10-like repeat during synthesis; the DNA-bound RNAP scrunches with synthesis to the end of the class II pause sequence. Either pathway results in the formation of the substrate for lambdoid phage Q proteins (4→6). Relaxation of the scrunched complex (4→5) yields a backtracked-like complex, in which the 3′ end of the RNA is extruded from the secondary channel but RNAP has not reverse translocated because σ70 – DNA contacts have been maintained. The scrunched complex can be regenerated when the backtracked-like complex is rescued by RNA cleavage, as above (5→2→4). Similar steps occur in σ70 region 4-dependent pausing, but with σ70 region 4 interacting with a –35-like repeat.
Review: σ70- dependent Transcription Pausing
as opposed to an obligatory release during the transition from initiation to elongation, is no longer in question36 but the distance at which σ70 can remain a part of elongation complexes and its effect on downstream elongation are unclear. Numerous structural and biochemical studies suggest that as RNA synthesis proceeds, σ70/RNAP contacts are disrupted, which should weaken the affinity of σ70 for core RNAP: namely, the σ70 region 3 linker that occupies the RNA exit channel should be displaced after synthesis of a few nucleotides and σ70 region 4 should be displaced after the RNA chain has lengthened to about 17 nt.25,37,38 Once the RNA is longer than 17 nt, it is expected that only σ70 regions 1.2 and 2 remain in contact with the core, although σ70 then competes with elongation factors such as NusG and NusA for these binding sites.39,40,41 Studies of early elongating RNAP complexes in vitro indicate that σ70 always remains associated with RNAP core at least as far as +16/+ 17 (likely to + 25). In one study, transcription was initiated on λpR′ with RNAP containing wild type σ70, σ70 L402F or σS, walked to + 16 via nucleotide deprivation, and then washed and restarted in the presence of a large molar excess of either the σ70 used for initiation or a competing σ70 with distinguishable pausing properties. In all cases, the pausing characteristics of the complexes were the same as those observed with the initiating σ70 without an exogenous σ70 challenge, suggesting that the same σ70 molecule directs both initiation and pausing.42 This experiment was not done with an 82pR′ template, the lambdoid phage late gene promoter for which pausing occurs at the greatest distance from + 1, although it is plausible that a similar result would be seen. FRET experiments on lacUV5 with a mutated – 10-like element (to eliminate the contribution of a σ70-retaining sequence) confirmed that up to 90% of complexes contain σ70 at least as far as + 15, and it is presumably the initiating σ70 that is present.5,43 These experiments further showed that 50–60% of complexes at + 50 contain σ70, but the question of retention versus rebinding was not addressed.43 Relative to the late gene promoters of lambdoid phages λ, 80 and 21, the 82pR′ pause occurs 7~9 nt further downstream. It appears to require a consensus extended –10 sequence (TnTGnTAnnnT) for full pause induction; if the underlined T in the extended – 10 sequence is mutated to G or C, + 25 pause efficiency is reduced ~ twofold (S.A.P. and J.W.R., unpublished results). In contrast, other late gene promoter pauses require only the AnnnT motif, suggesting that the further the –10-like sequence is from the + 1 start site the stronger a σ70-engaging sequence is required for paused complex formation. Consistently, if a series of spacers with lengths in the range from 2 to 20 nt is introduced to displace the λ + 16/+ 17 pause from + 19 up to + 37, only a weak pause remains unless an extended – 10 sequence is
789 present.2 When the local concentration of σ70 is increased via a translational fusion of σ70 to the β′ core subunit and is used to transcribe the λ template with a 20 nt spacer, a pause is observed at +37.29 Tethering σ70 to the core was shown to induce a σ70 region 2-dependent pause as far downstream as +462; additional experiments showed that a large molar excess of σ70 can also induce a pause at +462, suggesting that σ70 is no longer retained by the core at those distances from the +1 start site but it can reassociate with the core and display standard σ70 pause characteristics if present at a sufficiently high concentration.29 Several in vivo chromatin immunoprecipitation (ChIP) studies have been used to monitor promoterdistal σ70 retention throughout the genome. In these studies, proteins were chemically crosslinked to DNA and RNAP (the β or β′ subunit) or σ70 was precipitated with specific antibodies; the immunoprecipitated DNA was analyzed via PCR or microarray to identify the regions of the chromosome where these proteins are present. A study of ribosomal operons concluded that σ 70 travels downstream with RNAP and is released from the core stochastically;44 another ChIP-on-chip study of 109 transcription units found σ70 to be retained in the coding regions of nearly all promoters. 41 However, in other studies, retention was seen at only some promoters and only if the cells were grown in rich medium or were treated in the stationary phase.45,46 It is difficult to compare the results of these studies directly because the data were obtained from cells treated under differing growth conditions (mid-log versus stationary phase growth; rich versus minimal medium). Thus, the exact extent of in vivo downstream retention of σ70 is unclear, although these studies are consistent with some retention occurring, depending on the operon and growth conditions. Another caveat to using ChIP to assess σ70 retention is that σ70 makes basespecific contacts with promoter or pause DNA but it is not likely to make these contacts during elongation; thus, crosslinking efficiency might be greater at a promoter or sequence-encoded pause than downstream. Still, these experiments are beginning to shed light on σ70 retention during elongation and might reflect the effects of cellular factors that are absent from in vitro studies. An interesting hypothesis regarding σ70 retention is that one role of σ70-dependent promoter-proximal pausing is to retain σ70 in elongating complexes.5 A – 10-like element was introduced 20 nt downstream of the +16/+ 17 pause-inducing –10-like element in λpR′, and an appropriately displaced ~+ 37 pause was observed in vitro; however, the + 37 pause was much weaker if the upstream – 10-like element was mutated.47 This suggests that the +16/+ 17 pause can function to retain σ70 in the complex, essentially increasing its effective concentration downstream.
790 In fact, pause efficiency is increased at the downstream site to the same extent if an upstream – 10like element is present or if a ~ 30-fold increase in σ70 concentration is used. This study additionally showed via ChIP-PCR that σ70 retention is increased approximately twofold in vivo as far as ~ 700 nt downsteam, but again only if a promoter-proximal σ70-engaging –10-like element is present. σ70 retention was suggested in another study,6 in which the cspA and tnaA operons were shown to have several pauses in the first ~ 100 nt that are reduced in the presence of σ70 L402F, which strongly reduces σ70 region 2-dependent pausing. There are several candidate AnnnT sequences throughout the first ~ 100 nt that could function to retain σ70 for the next; however, mutational analysis of these – 10-like elements and their role in downstream pausing has not been conducted. In contrast, other studies of σ70 in elongation complexes suggest that promoter-proximal retention has no effect on promoter-distal retention. First, in the ChIP experiment noted above, there is no apparent downstream σ70 signal on the lacZYA operon under any growth conditions, despite the promoter-proximal pause identified later at this promoter.46 A single molecule FRET study of the lacUV5 promoter and its pause-deficient + 2G variant indicated that the wild type template initially retains more σ70 in complexes with less than 14 nt of RNA synthesized than do mutant templates but there is no difference between the templates in initial retention of σ70 in complexes with 50 nt of RNA, suggesting that upstream retention has no effect on downstream retention.43 Lastly, the efficiency of pausing for the 82pR′ σ70 region 4-dependent pause at + 14/+ 15 has no effect on the efficiency of pausing at the downstream + 25 region 2-dependent pause, although the former could plausibly have no affect on σ70 retention because pausing is induced by σ70 region 4 rather than region 2.3
Concluding remarks It is clear that σ70-dependent promoter-proximal pausing occurs during transcription from numerous promoters in E. coli. More distal σ70-dependent pausing can occur under special conditions in vitro, such as a higher local concentration of σ70, but it has not been shown clearly to occur in vivo. σ 70 dependent pausing requires only two sequence determinants: a repeat of either the promoter – 10 or the promoter – 35 element, and an appropriately spaced backtrack-inducing (class II pause) sequence; these minimal requirements suggest that pausing could occur throughout the E. coli genome and in other bacteria. With the exception of the lambdoid phage late gene promoters and the requirement of the pause for Q antitermination activity, the function
Review: σ70- dependent Transcription Pausing
of σ70-dependent pausing is unclear; it could equally well reflect accidental occurrence of pause-inducing sequences or an evolved regulatory function. Intriguingly, the mechanistically distinct eukaryotic RNA polymerase II promoter-proximal stalling appears also to require DNA-binding proteins, a related backtrack-inducing sequence and an RNA cleavage factor;7,9,48 these similarities suggest a generally important function of promoter-proximal pausing in transcription. Further studies are required to catalogue bacterial promoters that utilize σ 70 -dependent pausing and to determine any regulatory role it might have. In addition, characterizing the role of the backtrack-inducing sequence, especially the extent to which the G + C-rich and A + T-rich sequences affect scrunching and pause site determination, will be important in understanding the mechanism of σ70-dependent pausing.
Acknowledgements We thank Jeremy Bird for comments and the National Institutes of Health for support.
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