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Antitermination protein P7 of bacteriophage Xp10 distinguishes different types of transcriptional pausing by bacterial RNA polymerase
Journal Pre-proof Antitermination protein P7 of bacteriophage Xp10 distinguishes different types of transcriptional pausing by bacterial RNA polymerase Maria Prostova, Andrey Kulbachinskiy, Daria Esyunina PII:
S0300-9084(19)30382-7
DOI:
https://doi.org/10.1016/j.biochi.2019.12.011
Reference:
BIOCHI 5812
To appear in:
Biochimie
Received Date: 13 November 2019 Accepted Date: 23 December 2019
Please cite this article as: M. Prostova, A. Kulbachinskiy, D. Esyunina, Antitermination protein P7 of bacteriophage Xp10 distinguishes different types of transcriptional pausing by bacterial RNA polymerase, Biochimie, https://doi.org/10.1016/j.biochi.2019.12.011. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier B.V.
Abstract Bacteriophage-encoded transcription antiterminators play essential roles in the regulation of gene expression during infection. Here, we characterize the effects of the antiterminator protein P7 of bacteriophage Xp10 on transcriptional pausing by Xanthomonas oryzae RNA polymerase (RNAP) at different types of pause-inducing signals. When acting alone, P7 inhibits only hairpin-stabilized pauses, likely by preventing hairpin formation. In the presence of NusA, P7 also suppresses backtracking-stabilized pauses and the his elemental pause, but not the consensus elemental pause, suggesting that these pause signals may be mechanistically different. Thus, P7 and other bacteriophage proteins that bind near the RNA exit channel of RNAP have evolved to regulate transcription by suppressing RNAP pausing at a subset of regulatory signals, and to coopt NusA in doing so.
Antitermination protein P7 of bacteriophage Xp10 distinguishes different types of transcriptional pausing by bacterial RNA polymerase Maria Prostova, Andrey Kulbachinskiy*, Daria Esyunina* Institute of Molecular Genetics, Russian Academy of Sciences, Moscow 123182, Russia *
To whom correspondence should be addressed: Institute of Molecular Genetics, Russian Academy of Sciences Kurchatov sq. 2, Moscow 123182, Russia. Tel./Fax: +7 (499) 196 0015 Email: [email protected], [email protected]
Abstract Bacteriophage-encoded transcription antiterminators play essential roles in the regulation of gene expression during infection. Here, we characterize the effects of the antiterminator protein P7 of bacteriophage Xp10 on transcriptional pausing by Xanthomonas oryzae RNA polymerase (RNAP) at different types of pause-inducing signals. When acting alone, P7 inhibits only hairpin-stabilized pauses, likely by preventing hairpin formation. In the presence of NusA, P7 also suppresses backtracking-stabilized pauses and the his elemental pause, but not the consensus elemental pause, suggesting that these pause signals may be mechanistically different. Thus, P7 and other bacteriophage proteins that bind near the RNA exit channel of RNAP have evolved to regulate transcription by suppressing RNAP pausing at a subset of regulatory signals, and to coopt NusA in doing so.
Keywords: RNA polymerase / transcriptional pausing / NusA / transcription antitermination / bacteriophage
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1. Introduction During transcription, bacterial RNA polymerase (RNAP) reads multiple regulatory signals in the transcribed DNA and nascent RNA that allow it to identify promoters, synchronize transcription with translation, help to load specific transcription factors, and perform termination of RNA synthesis. Transcriptional pausing plays the central role in the regulation of transcription elongation and termination, and several types of pause-inducing signals were identified depending on the structural state of the paused transcription complex [1, 2]. The best characterized pause types include RNA hairpin-stabilized (represented by the his pause) and backtracking-stabilized pauses [1-5], with other examples of pre-translocated and posttranslocated pauses described in the literature [6, 7]. In particular, RNAP pausing at an oligoU sequence is the first step of intrinsic transcription termination, followed by RNA hairpin folding and transcription complex dissociation [8]. RNAP pausing during transcription initiation, dependent on RNAP-promoter contacts and clashing of the nascent RNA with the σ factor, was proposed to be a regulatory checkpoint that determines the efficiency of promoter escape [9-11]. Furthermore, analysis of RNAP pausing on the genomic scale resulted in the identification of a weakly conserved consensus pause sequence, which might modulate TEC interactions with regulatory factors and coordinate transcription with translation [12-14]. Independently of the pause type, it was proposed that most, if not all, pauses are offpathway states of the transcription elongation complex (TEC), which are formed through a common structural intermediate, the elemental pause [15]. The well characterized examples of the elemental paused complexes are TECs formed at the his pause signal in the absence of RNA hairpin and at the consensus pause site [1, 5, 12-14, 16-18]. The defining feature of these complexes is a half-translocated RNA-DNA hybrid, in which the RNA 3’-end adopts the posttranslocated conformation but the next template DNA nucleotide has not entered the active site and remains in the pre-translocated state thus inhibiting nucleotide addition; this is accompanied by subtle changes in the conformation of several RNAP elements in comparison with the active TEC [5, 17]. Furthermore, it was proposed that the elemental pause signal is multipartite, with contributions from the RNA-DNA hybrid, downstream and upstream fork-junctions at the transcription bubble, and downstream DNA, and that the TEC may sample different conformations in the elemental paused state, including half-translocated, pre-translocated and backtracked positions of the RNA-DNA hybrid [12-14, 17]. Thus, formation of the elemental paused complex may provide a time window required for its isomerisation to other types of paused complexes, including backward movement of RNAP during TEC backtracking [1]. However, possible functional differences between paused TECs formed at different variants of the elemental pause signal have remained unknown. Modulation of transcriptional pausing and termination by diverse factors plays multiple roles in the regulation gene expression, starting from classical mechanisms of transcription attenuation [19], to more recently discovered riboswitches [20, 21], and multiple cellular and bacteriophage-encoded noncoding RNAs and proteins with termination and antitermination activities [22-33]. Bacteriophages often encode small regulatory proteins that allow them to hijack the cellular transcription apparatus for their needs [22, 34]. Several of these proteins discovered over the course of last five decades – including N and Q of phage λ, gp39 of phage P23-45, and P7 of phage Xp10 – act as transcription antiterminators that allow transcription of downstream genes during different steps of phage infection [24, 26-30]. Bacteriophage-encoded antiterminator proteins were also shown to possess antipausing activities, but their effects on various classes of transcriptional pauses have not been systematically investigated [27-29]. Remarkably, all studied phage antiterminator factors bind near the RNA exit channel of RNAP, suggesting that they may in some way manipulate the nascent RNA structure and its interactions with RNAP [24, 26, 27, 29, 31, 35-37]. 2
P7 is a small 7 kDa protein encoded by bacteriophage Xp10 infecting Xanthomonas oryzae, an important rice pathogen [30, 38]. Previous studies demonstrated that in addition to transcription termination, P7 can also suppress transcriptional pausing, including hairpinstabilized pauses, but the molecular mechanisms of its antipausing activity remained only partially understood [29, 39]. Recent biochemical and structural analyses suggested that P7 can likely prevent formation of secondary RNA structures in the RNA exit channel of RNAP, acting together with the universal bacterial factor NusA [25, 29]. This may underlie its effects on intrinsic transcription termination and hairpin-stabilized pauses, which both depend on the formation of hairpins in nascent RNA. However, it remained unknown whether the antipausing activity of P7 strictly depended on hairpin folding or, alternatively, P7 could affect preceding steps in the pause formation, such as elemental pausing. In addition, it remained to be tested whether P7 could have any effects on other types of transcriptional pauses, including backtracking-stabilized pauses, and how these activities of P7 could be modulated by its cofactor NusA. To provide further insight into the mechanisms of P7 action, we analyzed its effects on various types of transcriptional pauses and demonstrated that its primary effect on hairpinstabilized pausing can be explained by the prevention of hairpin RNA folding. At the same time, NusA increases the spectrum of the pausing signals recognized by P7 and allows it to suppress backtracking-stabilized and some elemental pauses, but not the consensus pause. Therefore, the pathways of RNAP pausing appear to be different for different types of pause-inducing signals.
2. Materials and Methods 2.1. Proteins and nucleic acids X. oryzae core RNAP was purified from X. oryzae cells as described previously [29, 30]. The P7 protein of phage Xp10, the σA factor and NusA of X. oryzae were expressed and purified from E. coli [29]. E. coli GreA and GreB were obtained as described previously [29, 40]. DNA and RNA oligonucleotides used for TEC reconstitution were synthesized by DNA Synthesis and Evrogen (Moscow). 2.2. Transcription in vitro Analysis of hairpin-stabilized and consensus pausing was performed using synthetic oligonucleotide scaffolds containing 5′-labeled RNA (Fig. 1A and 2A), as described previously [13, 18, 29, 41]. Reconstituted TECs were incubated with 100 µM UTP, 100 µM CTP and 2 µM GTP (for hisP), or 100 µM CTP and 100 µM GTP (for consP) for increasing time intervals; chase reactions were performed with 1 mM NTPs after the final time point for additional 1 min. The reactions were stopped by the addition of an equal volume of EDTA/urea-containing stopbuffer, RNA products were analyzed by 19% denaturing PAGE and quantified by phosphorimaging using a Typhoon 9500 scanner (GE Healthcare). The efficiency of pausing was calculated as a ratio of the paused transcript to the sum of paused and read-through RNAs, with subtraction of residual pausing in the chase reactions. The data were fit to the single-exponential equation. Analysis of σ-dependent pausing was performed using synthetic oligonucleotide scaffold (Fig. 3A) as described in ref. [40, 42]. Briefly, reconstituted TECs containing 20 nM of 5′labeled RNA, 40 nM of the template DNA strand, 200 nM of the nontemplate DNA strand and 40 nM of X. oryzae core RNAP were supplied with 1 µM σA subunit, 1.5 µM P7 and/or 0.7 µM NusA; GreB was added to 1 µM when indicated. The TECs were incubated with all four NTPs (100 µM each) at 30°C for increasing time intervals, followed by analysis of RNA products by 3
PAGE 19% and phosphorimaging. The relative pause efficiency was calculated as the ratio of the paused RNA products (24/25 nt) in each sample to the amount of paused RNA at the first time point in reactions containing the σA subunit nut lacking other factors. Analysis of oligoU-dependent pausing was performed on a PCR DNA template containing the U-tract from the λ tR2 terminator placed downstream of the λ PR promoter with a 26-mer Cless initially transcribed sequence (Fig. 4A) as described previously [29]. 26-mer TECs containing radiolabeled RNA were obtained by performing transcription with limited substrate set (25 µM ApU, 100 µM ATP, 100 µM GTP with addition of α-[32P]-UTP), followed by the addition of P7 (5 µM), GreA (2 µM), and/or NusA (0.5 µM) and all four NTPs (100 µM ATP, GTP, CTP, 25 µM UTP) at 30 °C.
3. Results 3.1. Distinct effects of P7 and NusA on hairpin-stabilized and elemental pausing at the his pause signal To assess the role of RNA folding in the antipausing activity of P7, we analyzed hairpinstabilized pausing by X. oryzae RNAP using reconstituted TECs, in which RNA hairpin formation could be mimicked by the addition of antisense RNA. The TECs were reconstituted from the core RNAP and synthetic oligonucleotides, based on the sequence of the his pause (hisP) signal from the histidine biosynthesis operon of enterobacteria (Fig. 1A). In the absence of RNA hairpin, this sequence was shown to induce elemental pausing, depending on RNAP interactions with the RNA-DNA hybrid, upstream and downstream forks and downstream DNA duplex [1, 17]. In the presence of RNA hairpin, such complexes recapitulate the properties of paused TECs containing full RNA hairpin obtained during promoter-dependent transcription in vitro [18]. The RNA oligonucleotide used for TEC reconstitution did not contain hairpin, thus allowing to analyze elemental pausing. The hairpin formation was mimicked by the addition of a short antisense RNA oligonucleotide (asRNA) complementary to the upstream part of the RNA transcript (Fig. 1A). We first tested whether P7 could suppress transcriptional pausing in reconstituted complexes. In agreement with its reported effects on hairpin-stabilized pausing [29], P7 decreased pausing in TECs reconstituted in the presence of asRNA, confirming that these complexes can be used to study the antipausing activity of P7: the pause half-life time (t1/2) was decreased 3-fold, from 61.8 ± 2.2 s in the absence of P7 to 20.2 ± 2.7 s in its presence (Fig. 1B and C, filled symbols). As expected, the pause half-life was significantly decreased in the absence of asRNA (t1/2 = 15 ± 1.2 s). Strikingly, in this case, P7 had no effect on RNAP pausing or even slightly increased it (t1/2 = 17.8 ± 1.6 s; Fig. 1B and C, open symbols). Furthermore, in the presence of P7 the pause half-lives were identical independently of the asRNA addition (17.8 ± 1.6 s vs. 20.2 ± 2.7 s, Fig. 1B), suggesting that P7 likely acts by preventing RNA duplex formation in the RNA exit channel of RNAP, in agreement with structural data [25]. We next analyzed the effects of P7 on pausing in the presence of NusA. NusA normally stimulates the recognition of pausing and termination signals by bacterial RNAP but can be recruited by phage-encoded proteins, including P7, to act as a cofactor during antitermination [1, 22, 43]. It was previously demonstrated that NusA stimulates the antipausing activity of P7 and also increases its apparent affinity to RNAP [29]. We showed that X. oryzae NusA indeed increased pausing in reconstituted TECs containing asRNA in the absence of P7 (t1/2 of 84.2 ± 5.0 s). In agreement with published data [18], this increase was modest in comparison with TECs containing longer RNA duplexes or full his RNA hairpin, but significant. In contrast, 4
NusA stimulated the antipauing effect of P7 in comparison with NusA-less complex (t1/2 of 11.3 ± 2.0 s vs. 20.2 ± 2.7 s in the absence of NusA) (Fig. 1D). NusA had no strong effect on elemental pausing in hairpin-less TEC, reconstituted in the absence of asRNA (t1/2 of 21.1 ± 1.9 s in comparison with 15 ± 1.2 s without NusA). However, P7 together with NusA suppressed pausing in the absence of asRNA to the same level as in its presence (t1/2 of 10.2 ± 2.0 s vs. 11.3 ± 2.0 s). Furthermore, the half-life time of elemental pausing (determined in the absence of asRNA) in the presence of both P7 and NusA was significantly (p<0.05) smaller than the half-life time measured in their absence (t1/2 of 10.2 ± 2.0 s vs. 15.0 ± 1.2 s, Fig. 1B and C) demonstrating that both proteins cooperate to suppress elemental pausing at the hisP signal independently of RNA hairpin formation. 3.2. P7 and NusA do not suppress consensus pausing The consensus pause was discovered previously in the whole-genome NET-seq experiments in E. coli, resulting in the identification of a widespread pausing signal, the most conserved features of which are the -1C/T, +1G motif near the RNA 3’-end, and the -11G, -10G dinucleotide at the upstream border of the RNA/DNA hybrid (Fig. 2A) [12-14]. A similar pause sequence was identified in B. subtilis [13]. Further analysis demonstrated that it has a multipartite nature and likely stabilizes the TEC in the half-translocated conformation, similarly to the hisP signal. It was therefore defined as the consensus elemental pause signal (consP), likely underlying most pausing events [17] (see Introduction). We previously showed that, in contrast to hairpinstabilized pausing, P7 could not suppress consensus pausing or even slightly increased its duration [29]. Since the experiments presented in Fig. 1 demonstrated that NusA can assist P7 to suppress elemental pausing, we tested whether it could also help P7 to prevent pausing at the consensus pause site. Analysis of consensus pausing was performed in reconstituted TECs containing the previously established consensus pause sequence (Fig. 2A) [13]. In accordance with published data, P7 did not affect or even slightly increased the pause half-life (t1/2 of 16.6 ± 3.2 s vs. 22.5 ± 4.3 s, Fig. 2B and C; see also Fig. S1 below) [29]. It should be noted that in this case transcription was performed at higher NTP concentrations than in the case of his elemental pause, because consP is a stronger pause signal with a longer half-life time, e.g. [1, 16, 17]. Strikingly, NusA did not have any significant effect on pausing both in the absence and in the presence of P7 (t1/2 of 14 s and 21 s,respectively; Fig. 2B and D). Therefore, the consensus pause is functionally different from the his elemental pause present in the hisP signal, in that it cannot be suppressed by simultaneous action of P7 and NusA. Since P7 and NusA bind near the RNA exit channel of RNAP, we tested whether the synthetic oligoU sequence present in the RNA 5’-end in the consP complexes could affect formation of paused complexes and their sensitivity to P7 and NusA, by analyzing a modified version of the consP scaffold containing the 5’-RNA sequence from the hisP complex (Fig. S1A). It was found that this modification did not strongly change the pause half-life times either in the absence or in the presence of P7 or NusA in comparison with the original scaffold, and P7 together with NusA even increased the pause duration (Fig. S1B). 3.3. P7 and NusA cooperate to suppress backtracking-stabilized pausing TEC backtracking can lead to prolonged RNAP stalling or permanent arrest of transcription [4446]. Backtracked TECs can be reactivated by transcript cleavage factors, GreA in most bacteria, GreA or GreB in E. coli and X. oryzae, or TFIIS in the case of eukaryotic RNAP II. Several 5
previously characterized pause signals were shown to be associated with backtracking, including σ-dependent pauses [40, 47-49], oligoU sequences [29, 31] and ops sites [3, 50], as revealed by their suppression by Gre factors. The consensus paused complex can also readily adop a backtracked conformation sensitive to Gre-induced RNA cleavage, but this does not seem to contribute to the pause half-life [13, 17]. To reveal whether P7 and NusA can affect RNAP backtracking, we tested their action on the formation of backtracking-stabilized pauses of various types by X. oryzae RNAP. The existence of σ-dependent pausing in X. oryzae has never been studied. To reveal whether the σA factor of X. oryzae can induce pausing by its RNAP, we reconstituted TEC on a synthetic template containing the consensus σ-dependent pause signal (TGnTATAAT, where ‘n’ is any nucleotide), which was used previously to analyze σ-dependent pausing by RNAPs from various groups of bacteria [40, 42, 51, 52] (Fig. 3A). The expected pause position was located 35 nucleotides downstream of the 3’-end of the starting RNA transcript, corresponding to the site of pausing previously observed for the σ70- and σA-dependent pauses for E. coli, Thermus aquaticus and Deinococcus radiodurans RNAPs [40, 42, 47, 49, 51]. We found that in the presence of X. oryzae σA transcription was efficiently stalled at the expected pause site (24-26 nt RNAs), with pause half-life exceeding 5 minutes (Fig. 3B and C, compare panels 1 and 2). GreB decreased the efficiency of pausing at positions 24/25 and fully suppressed pausing at position 26 (Fig. 3B and C, panel 6), confirming that the paused complexes adopt a backtracked conformation and can be reactivated by RNA cleavage [40, 42]. P7 or NusA alone had only small effects on σ-dependent pausing (panels 3 and 4). However, when present together, P7 and NusA significantly reduced the efficiency of pausing (to 55-60%, p<0.01) (panel 5). At the same time, P7 plus NusA did not decrease the duration of pausing (little pause escape was observed at the 5 minute reaction point), suggesting that they suppress initial pause formation rather than decrease the pause dwell time. The ops sequence is a pause-inducing signal located in promoter-proximal regions of certain operons in E. coli and other proteobacteria and required for recruitment of an antitermination factor RfaH for efficient transcription of downstream genes. The 12 nt ops signal can be specifically recognized by RNAP and RfaH in the nontemplate DNA strand and induce limited backtracking (Fig. S2A) [3, 50, 53]. Previously, it was shown that P7 can suppress pausing at the ops sequence [39], although the antipausing effect might be milder than for hairpin-dependent pausing and termination signals [29, 39]. We tested whether NusA could enhance the antipausing activity of P7 on an ops-containing template. Interestingly, under conditions of our experiments P7 itself had only small if any effect on ops-dependent pausing by X. oryzae RNAP. At the same, the longevity of ops pause was strongly decreased in the presence of both P7 and NusA (Fig. S2B). OligoU-motifs represent another type of a pause-inducing signal that is associated with TEC backtracking. In the context of intrinsic terminators, the oligoU sequence first induces transient pausing by RNAP, followed by RNA hairpin folding that further destabilizes the TEC and leads to its dissociation [8]. During termination the RNA hairpin formation likely prevents backward RNAP translocation, but isolated oligoU-motifs can promote TEC backtracking, likely due to unstable RNA/DNA hybrid formed in the RNAP active center [29, 31]. In agreement with our previous report [29], X. oryzae RNAP paused at several sites at the oligoU sequence, taken from the λ tR2 terminator and placed downstream of a 26-mer C-less sequence under the control of the λ PR promoter (Fig. 4A; panel 1 in Fig. 4B). RNAP stalling at these sites was suppressed by GreA, confirming the formation of backtracked TECs during transcription (Fig. 4B, panel 3). P7 had no significant effect on pausing, when present either alone or together with GreA (Fig. 4B, compare panels 1, 2 and 3, 4). Thus, P7 by itself cannot prevent RNAP backtracking and 6
does not cooperate with GreA in reactivation of backtracked TECs. At the same time, P7 together with NusA suppressed oligoU-dependent pausing (Fig. 4B, panel 6), in accordance with published data [29], while NusA alone stimulated pausing (panel 5). The above results demonstrated that P7 and NusA can suppress different types of backtracking-stabilized pauses. To reveal whether P7 and NusA might also directly reactivate backtracked TECs, we analyzed their effects on RNAP activity in reconstituted TEC containing a mismatched nucleotide at the RNA 3’-end (Fig. S3A). The mismatch promotes one-nucleotide backtracking of the TEC with subsequent cleavage of the 3’-dinucleotide from the RNA transcript, resulting from the intrinsic RNA cleavage activity of RNAP [54, 55]. To avoid premature RNA cleavage during TEC assembly, the complex was obtained in the absence of Mg2+ ions. Incubation of the reconstituted TEC with NTP substrates and Mg2+ led to both extension and cleavage of the starting RNA transcript (Fig. S3B). Because of the presence of mismatch, the kinetics of RNA extension in this complex was much slower than in the case of fully-matched complexes (e.g. [54]). NusA and P7 did not have an inhibitory effect on RNA cleavage or a stimulatory effect on the extension of the mismatched RNA by X. oryzae RNAP (Fig. S5B and S5C). In fact, the kinetics of RNA extension in the presence of both P7 and NusA was even slower than in the absence of factors (Fig. S5C). This result suggested that P7 and NusA cannot reactivate preformed backtracked TECs.
4. Discussion 4.1. Antitermination and antipausing activities of P7 Transcription antitermination is an essential process in the regulation of bacterial and bacteriophage gene expression [22]. Previous studies identified several cellular and phageencoded regulatory proteins that act as transcription antiterminators, but for most of them the molecular mechanisms of action have remained only partially understood [24-30]. Here, we analyzed the antipausing activity of the P7 antiterminator protein encoded by phage Xp10 of X. oryzae and revealed that it has distinct effects on various types of transcriptional pauses. Thus, this small regulatory protein can discriminate between different functional states of the TEC, likely underlying its specific effects on transcription termination. Previous studies suggested that various types of transcriptional pauses, including those studied in this work, are formed through the same intermediate, the elemental pause [1, 16, 17]. The two best studied elemental pause signals, hisP (without RNA hairpin) and consP, have several common nucleotides at key positions (-10, -1 and +1; Fig. 1A and Fig. 2A), suggesting that both complexes may be functionally similar. Their analysis demonstrated that the elemental paused complex can isomerize between the preferable half-translocated and less-populated pretranslocated or backtracked conformations. Changes in various parts of the pause signal may possibly affect the equilibrium between these states and change the functional properties of the paused complex [1, 17]. During hairpin-stabilized pausing, the RNA hairpin formation stabilizes the half-translocated TEC conformation and induces ‘swiveling’ of several domains of the RNAP β′ subunit, including clamp, shelf, jaw and the SI3 insertion in the trigger loop in the active center, around an axis roughly coinciding with the bridge helix of RNAP (Fig. 5A) [4, 16]. The swiveled conformation inhibits trigger loop folding by changing the position of the SI3 insertion, further inhibiting nucleotide addition. The universal bacterial factor NusA stimulates hairpin-stabilized, but not elemental, pausing by binding near the RNA exit channel and directly interacting with the flap domain of RNAP and nascent RNA, thus promoting hairpin formation (Fig. 5A) [4, 16, 43]. 7
We showed that P7 specifically suppresses hairpin-stabilized pausing by bacterial RNAP, likely by preventing RNA folding in the RNA exit channel of RNAP. Indeed, the duration of the his pause in the presence of P7 was decreased to the level of the hairpin-less elemental pause with the same sequence, which by itself was not affected by P7. These observations are supported by the recently published structure of TEC of X. oryzae RNAP with bound P7 [25]. In this structure, the RNA exit channel between the β flap domain and the main RNAP body is constricted by P7 and is too narrow for RNA hairpin formation (Fig. 5B). We showed that the activity of P7 at the hisP signal and its affinity to the TEC are enhanced by NusA ([29] and this work). As revealed in the TEC structure, P7 binding is directly stimulated by NusA that interacts with P7 at the RNA exit channel (Fig. 5C) [25]. Together, P7 and NusA may stabilize the closed conformation of the TEC and likely prevent RNAP swiveling, thus explaining the stimulatory effects of NusA on the antipausing activity of P7. In contrast to hairpin-stabilized pausing and intrinsic termination, P7 does not significantly affect other types of analyzed pauses, including his elemental and consensus elemental pauses, as well as backtracking-associated oligoU-dependent and σ-dependent pauses. This suggests that P7 likely cannot prevent conformational changes of the TEC associated with formation of these pauses. In comparison, NusA is also unable to regulate transcriptional pauses which are not associated with RNA hairpin formation in the RNA exit channel [3, 18, 43]. Strikingly, however, P7 in the presence of NusA can suppress the his elemental pause and various types of backtracking-stabilized pauses, including σ-dependent and ops pauses which also contain the elemental pause motif, in particular, G at position +1 (Fig. 3, Fig. S2) [56, 57]. Thus, when P7 and NusA are simultaneously bound to these TECs they might counteract elemental pausing associated with isomerization of the TEC into the inactive half-translocated conformation. P7 was shown to promote forward translocation of the TEC [39]; while this activity is apparently insufficient for suppression of elemental and backtracking-stabilized pausing by P7 alone, it may be further enhanced by NusA. Furthermore, since both factors bind at the upstream part of the swiveling module of RNAP, they might physically prevent its rotation that could transiently occur during elemental pausing (Fig. 5C) [1, 16, 17]. At the same time, these factors cannot apparently reactivate already backtracked complexes, as demonstrated by the absence of their effect on the longevity of σ-dependent pauses (Fig. 3) or on RNAP activity in backtracked mismatched complexes (Fig. S3). Recent structural analysis of a backtracked TEC revealed that it mostly adopts a swiveled conformation, similar to the elemental paused complex [4, 16, 58]. However, the location of the RNA 3’-end in the secondary channel as a result of backward TEC translocation likely makes impossible direct activation of such complexes by P7 and NusA, even if these factors could reverse RNAP swiveling. Intriguingly, the consensus elemental pause, which acts as a universal pause signal in various bacteria and even eukaryotes [13], is resistant to the action of P7 and NusA, in contrast to the his elemental pause and backtracking-stabilized pauses containing the elemental pause motifs. This result confirms that the consensus pause signal has a multipartite nature and the weekly conserved positions in this motif, identified in refs. [12, 13] (shown in plain letters in Fig. 2A), contribute to its recognition by RNAP. As a result, the TEC formed at the consensus pause site may be either more strongly stabilized in the same conformation as in the case of the his elemental pause (due to stronger RNAP interactions with the consensus sequence), or adopt another conformation different from the his elemental pause, thus making it insensitive to P7 and NusA. Therefore, although both complexes were proposed to fluctuate between different translocation states [12, 16, 17, 59], they may have different preferable conformations resulting in their different sensitivity to the antipausing action of P7 and NusA.
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4.2. Antipausing activities of various antitermination factors Analysis of bacteriophage antiterminator proteins reveals that, in addition to antitermination, all these factors can suppress various types of transcriptional pauses. Most studied antiterminators, including P7 of phage Xp10, N and Q of λ, gp39 of P23-45, and put RNA of HK022, bind at different sites around the RNA exit channel of RNAP (Fig. 5B,C). Accordingly, N [27], Q [26] and P7 (this work and [25]) were shown to prevent RNA folding in the RNA exit channel and to protect nascent RNA from the action of pro-termination factors. Recent structural analysis of these three proteins, N, Q and P7, confirmed that they interfere with RNA hairpin formation, thus explaining their effects on intrinsic transcription termination and hairpin-stabilized pausing [24, 25, 27]. At the same time, these antiterminator proteins rely on different sets of RNA/RNAP interactions for their antipausing/antitermination activities. Both N and P7 recruit NusA to rearrange the RNA exit channel; however, while the former protein destabilizes RNA hairpin formation by moving NusA and the flap domain of RNAP away from nascent RNA, the latter one constricts the RNA exit channel and likely redirects the transcript through an alternative way out of RNAP [25, 27]. In contrast, analysis of the Q protein from the lambdoid phage 21 demonstrated that it forms a narrow ring on nascent RNA at the exit from RNAP, thus effectively shielding it from secondary structure formation or termination factor binding [24, 60]. It remains to be known whether NusA can stimulate the antipausing activity of Q21 and its loading onto RNA, similarly to previously studied proteins Qλ and Q82 [26, 61, 62]. Different groups of antitermination factors may target common intermediates in the pausing pathway even if they bind at different sites of RNAP. P7 and other bacteriophage proteins mentioned above prevent RNA folding by changing the conformation of the RNA exit channel and restricting RNAP opening, thus stabilizing the active state of the TEC. The bacterial antiterminator RfaH acts in a similar way, while binding at the upstream part of the DNA binding cleft of RNAP [5]. RfaH was also shown to prevent RNAP swiveling [5], similarly to the proposed cooperative action of P7 and NusA. Further analysis of the antipausing and antitermination activities of small phage-encoded protein regulators may provide deeper insights into the intricate conformational mobility of the TEC, help to discover new antipausing mechanisms and develop robust synthetic switches for the regulation of gene expression in bacteria.
Authors’ contributions AK and DE conceived the study and designed experiments, MP and DE performed experiments and analyzed data. AK wrote the manuscript. All authors read and approved the manuscript.
Conflict of Interest The authors declare that there is no conflict of interest.
Acknowledgements We thank Dr. Ivan Petushkov for useful discussions, I. Artsimovitch for plasmids.
Funding This work was supported in part by the Russian Science Foundation (grant 17-14-01393 to AK, analysis of transcription antitermination) and Russian Foundation for Basic Research (grant 1834-20095 to DE, analysis of σ-dependent pausing).
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[57] E.J. Strobel, J.W. Roberts, Two transcription pause elements underlie a sigma70-dependent pause cycle, Proc Natl Acad Sci U S A, 112 (2015) E4374-4380. [58] M. Abdelkareem, C. Saint-Andre, M. Takacs, G. Papai, C. Crucifix, X. Guo, J. Ortiz, A. Weixlbaumer, Structural Basis of Transcription: RNA Polymerase Backtracking and Its Reactivation, Mol Cell, 75 (2019) 298-309 e294. [59] P.P. Hein, K.E. Kolb, T. Windgassen, M.J. Bellecourt, S.A. Darst, R.A. Mooney, R. Landick, RNA polymerase pausing and nascent-RNA structure formation are linked through clamp-domain movement, Nat Struct Mol Biol, 21 (2014) 794-802. [60] Z. Yin, J.T. Kaelber, R.H. Ebright, Structural basis of Q-dependent antitermination, Proc Natl Acad Sci U S A, 116 (2019) 18384-18390. [61] C.D. Wells, P. Deighan, M. Brigham, A. Hochschild, Nascent RNA length dictates opposing effects of NusA on antitermination, Nucleic Acids Res, 44 (2016) 5378-5389. [62] X.J. Yang, J.A. Goliger, J.W. Roberts, Specificity and mechanism of antitermination by Q proteins of bacteriophages lambda and 82, J Mol Biol, 210 (1989) 453-460. [63] M. Turtola, G.A. Belogurov, NusG inhibits RNA polymerase backtracking by stabilizing the minimal transcription bubble, eLife, 5 (2016).
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Figure legends Figure 1. Effects of P7 and NusA on hairpin-stabilized and elemental pausing at the his pause site. (A) Scheme of the nucleic acid scaffold based on the hisP sequence and used for TEC assembly; DNA is black RNA is red. The TECs were reconstituted from synthetic oligonucleotides either with or without addition of asRNA, followed by addition of P7 and/or NusA and NTPs (CTP, UTP, GTP), resulting in the extension of the starting 17 nt RNA by four nucleotides. The 19 nt paused RNA is indicated with an arrow. Nucleotides corresponding to the consensus pause sequence are shown in bold (see Fig. 2A) [12, 13]. Nucleotide positions relative to the pause sites are numbered above the scheme. (B) Analysis of the kinetics of RNA extension in the absence of NusA. Chase reactions (‘C’) were performed with the addition of high concentrations of NTPs after the final time point. Positions of the starting 17 nt, paused 19 nt and readthrough 21 nt RNAs are indicated. Note that a fraction of the starting RNA was not extended likely because it was not bound by RNAP during TEC reconstitution. (C) Kinetics of pausing. At each time point, the efficiency of pausing was quantified as the ratio of paused to the sum of paused and readthrough RNAs, with subtraction of paused RNAs in the chase reaction (‘C’). The pause half-life times are shown on the right (means and standard deviations from three independent experiments). Figure 2. Effects of P7 and NusA on pausing at the consP pause site. (A) Scheme of the nucleic acid scaffold used for TEC assembly. The consensus pause sequence (‘Cons’) is shown above the scheme; the most conserved positions are bold [12, 13]; nucleotides are numbered relative to the pause position. The TECs were reconstituted from synthetic oligonucleotides, followed by the addition of P7 and/or NusA and NTPs (GTP, CTP), resulting in the extension of the starting 15 nt RNA by seven nucleotides. The 17 nt paused RNA is indicated with an arrow. (B) Analysis of RNA extension in the absence and in the presence of P7 and/or NusA. Positions of the starting 15 nt, paused 17 nt and readthrough 22 nt RNAs are indicated. The unextended fraction of 15 nt RNA corresponds to RNA that was not bound by RNAP during TEC reconstitution. (The samples in NusA-containing reactions were separated on the same gel, and a part of the gel was cut off to remove empty lanes). (C) Kinetics of pausing. The pause half-life times are shown on the right (means and standard deviations from three independent experiments). Figure 3. Analysis of σ-dependent pausing by X. oryzae RNAP. (A) Scheme of the nucleic acid scaffold used for TEC assembly. The TECs were reconstituted from synthetic oligonucleotides, followed by addition of the σA subunit, P7 and/or NusA and all four NTPs. Positions of the -10-like element, starting 20 nt and paused 25 nt RNAs are indicated. Nucleotides corresponding to the consensus pause sequence are shown in bold (positions -11, -1 and +1; see Fig. 2A) (B) Analysis of σ pause formation in the absence and in the presence of P7, NusA and GreA. Positions of the starting 20 nt, paused 25 nt and run-off (RO) RNAs are indicated. The panel numbers are indicated under the gel. (C) Quantification of pause efficiencies. For each reaction point, the efficiency of pausing at positions 24/25 is shown relative to pausing measured at the 0.5 min time point in the presence of σA and in the absence of other factors (means and standard deviations from three independent experiments). Figure 4. Analysis of oligoU-dependent pausing by X. oryzae RNAP. (A) The initially transcribed sequence of the oligoU template. The +1 and +26 positions are indicated, the oligoUmotif from the λ tR2 terminator is shown in pink, the 31-37 nt pausing region is shown with blue line. (B) PAGE-analysis of RNA products synthesized on the oligoU template. Positions of the starting 26-mer RNA, 31-37 nt paused RNA products and full-length run-off (RO) RNA are indicated. The chase reactions (‘C’, last lane in each panel) were performed with high 14
concentrations of NTPs (1 mM each) added after the 10’ time point. The panel numbers are indicated under gel. Figure 5. Structural models of paused and P7-bound TECs. (A) Hairpin-containing E. coli TEC with bound NusA at the hisP pause signal. The direction of RNAP swiveling in the paused complex in comparison with active TEC is shown with a blue arrow. (B) TEC of X. oryzae RNAP with bound P7 (shown as a light blue surface). (C) TEC of X. oryzae RNAP with bound P7 and NusA; the narrow RNA exit channel between the Zn-binding domain (ZBD), P7 and β flap is shown with an arrow. The RNA transcript is yellow, the flap domain in the RNAP β subunit is red, the ZBD in the β’ subunit is brownish, NusA is light green. The N-terminus of the β’ subunit involved in P7 binding is black. The PDB accession numbers for each structure are indicated.
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Highlights: • • • •
Small antiterminator protein P7 of phage Xp10 modulates pausing by RNA polymerase P7 prevents RNA duplex formation during hairpin-stabilized pausing P7 cooperates with NusA to suppress elemental and backtracking-stabilized pauses The consensus elemental pause is resistant to the action of P7 and NusA
S0300-9084(19)30382-7
DOI:
https://doi.org/10.1016/j.biochi.2019.12.011
Reference:
BIOCHI 5812
To appear in:
Biochimie
Received Date: 13 November 2019 Accepted Date: 23 December 2019
Please cite this article as: M. Prostova, A. Kulbachinskiy, D. Esyunina, Antitermination protein P7 of bacteriophage Xp10 distinguishes different types of transcriptional pausing by bacterial RNA polymerase, Biochimie, https://doi.org/10.1016/j.biochi.2019.12.011. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier B.V.
Abstract Bacteriophage-encoded transcription antiterminators play essential roles in the regulation of gene expression during infection. Here, we characterize the effects of the antiterminator protein P7 of bacteriophage Xp10 on transcriptional pausing by Xanthomonas oryzae RNA polymerase (RNAP) at different types of pause-inducing signals. When acting alone, P7 inhibits only hairpin-stabilized pauses, likely by preventing hairpin formation. In the presence of NusA, P7 also suppresses backtracking-stabilized pauses and the his elemental pause, but not the consensus elemental pause, suggesting that these pause signals may be mechanistically different. Thus, P7 and other bacteriophage proteins that bind near the RNA exit channel of RNAP have evolved to regulate transcription by suppressing RNAP pausing at a subset of regulatory signals, and to coopt NusA in doing so.
Antitermination protein P7 of bacteriophage Xp10 distinguishes different types of transcriptional pausing by bacterial RNA polymerase Maria Prostova, Andrey Kulbachinskiy*, Daria Esyunina* Institute of Molecular Genetics, Russian Academy of Sciences, Moscow 123182, Russia *
To whom correspondence should be addressed: Institute of Molecular Genetics, Russian Academy of Sciences Kurchatov sq. 2, Moscow 123182, Russia. Tel./Fax: +7 (499) 196 0015 Email: [email protected], [email protected]
Abstract Bacteriophage-encoded transcription antiterminators play essential roles in the regulation of gene expression during infection. Here, we characterize the effects of the antiterminator protein P7 of bacteriophage Xp10 on transcriptional pausing by Xanthomonas oryzae RNA polymerase (RNAP) at different types of pause-inducing signals. When acting alone, P7 inhibits only hairpin-stabilized pauses, likely by preventing hairpin formation. In the presence of NusA, P7 also suppresses backtracking-stabilized pauses and the his elemental pause, but not the consensus elemental pause, suggesting that these pause signals may be mechanistically different. Thus, P7 and other bacteriophage proteins that bind near the RNA exit channel of RNAP have evolved to regulate transcription by suppressing RNAP pausing at a subset of regulatory signals, and to coopt NusA in doing so.
Keywords: RNA polymerase / transcriptional pausing / NusA / transcription antitermination / bacteriophage
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1. Introduction During transcription, bacterial RNA polymerase (RNAP) reads multiple regulatory signals in the transcribed DNA and nascent RNA that allow it to identify promoters, synchronize transcription with translation, help to load specific transcription factors, and perform termination of RNA synthesis. Transcriptional pausing plays the central role in the regulation of transcription elongation and termination, and several types of pause-inducing signals were identified depending on the structural state of the paused transcription complex [1, 2]. The best characterized pause types include RNA hairpin-stabilized (represented by the his pause) and backtracking-stabilized pauses [1-5], with other examples of pre-translocated and posttranslocated pauses described in the literature [6, 7]. In particular, RNAP pausing at an oligoU sequence is the first step of intrinsic transcription termination, followed by RNA hairpin folding and transcription complex dissociation [8]. RNAP pausing during transcription initiation, dependent on RNAP-promoter contacts and clashing of the nascent RNA with the σ factor, was proposed to be a regulatory checkpoint that determines the efficiency of promoter escape [9-11]. Furthermore, analysis of RNAP pausing on the genomic scale resulted in the identification of a weakly conserved consensus pause sequence, which might modulate TEC interactions with regulatory factors and coordinate transcription with translation [12-14]. Independently of the pause type, it was proposed that most, if not all, pauses are offpathway states of the transcription elongation complex (TEC), which are formed through a common structural intermediate, the elemental pause [15]. The well characterized examples of the elemental paused complexes are TECs formed at the his pause signal in the absence of RNA hairpin and at the consensus pause site [1, 5, 12-14, 16-18]. The defining feature of these complexes is a half-translocated RNA-DNA hybrid, in which the RNA 3’-end adopts the posttranslocated conformation but the next template DNA nucleotide has not entered the active site and remains in the pre-translocated state thus inhibiting nucleotide addition; this is accompanied by subtle changes in the conformation of several RNAP elements in comparison with the active TEC [5, 17]. Furthermore, it was proposed that the elemental pause signal is multipartite, with contributions from the RNA-DNA hybrid, downstream and upstream fork-junctions at the transcription bubble, and downstream DNA, and that the TEC may sample different conformations in the elemental paused state, including half-translocated, pre-translocated and backtracked positions of the RNA-DNA hybrid [12-14, 17]. Thus, formation of the elemental paused complex may provide a time window required for its isomerisation to other types of paused complexes, including backward movement of RNAP during TEC backtracking [1]. However, possible functional differences between paused TECs formed at different variants of the elemental pause signal have remained unknown. Modulation of transcriptional pausing and termination by diverse factors plays multiple roles in the regulation gene expression, starting from classical mechanisms of transcription attenuation [19], to more recently discovered riboswitches [20, 21], and multiple cellular and bacteriophage-encoded noncoding RNAs and proteins with termination and antitermination activities [22-33]. Bacteriophages often encode small regulatory proteins that allow them to hijack the cellular transcription apparatus for their needs [22, 34]. Several of these proteins discovered over the course of last five decades – including N and Q of phage λ, gp39 of phage P23-45, and P7 of phage Xp10 – act as transcription antiterminators that allow transcription of downstream genes during different steps of phage infection [24, 26-30]. Bacteriophage-encoded antiterminator proteins were also shown to possess antipausing activities, but their effects on various classes of transcriptional pauses have not been systematically investigated [27-29]. Remarkably, all studied phage antiterminator factors bind near the RNA exit channel of RNAP, suggesting that they may in some way manipulate the nascent RNA structure and its interactions with RNAP [24, 26, 27, 29, 31, 35-37]. 2
P7 is a small 7 kDa protein encoded by bacteriophage Xp10 infecting Xanthomonas oryzae, an important rice pathogen [30, 38]. Previous studies demonstrated that in addition to transcription termination, P7 can also suppress transcriptional pausing, including hairpinstabilized pauses, but the molecular mechanisms of its antipausing activity remained only partially understood [29, 39]. Recent biochemical and structural analyses suggested that P7 can likely prevent formation of secondary RNA structures in the RNA exit channel of RNAP, acting together with the universal bacterial factor NusA [25, 29]. This may underlie its effects on intrinsic transcription termination and hairpin-stabilized pauses, which both depend on the formation of hairpins in nascent RNA. However, it remained unknown whether the antipausing activity of P7 strictly depended on hairpin folding or, alternatively, P7 could affect preceding steps in the pause formation, such as elemental pausing. In addition, it remained to be tested whether P7 could have any effects on other types of transcriptional pauses, including backtracking-stabilized pauses, and how these activities of P7 could be modulated by its cofactor NusA. To provide further insight into the mechanisms of P7 action, we analyzed its effects on various types of transcriptional pauses and demonstrated that its primary effect on hairpinstabilized pausing can be explained by the prevention of hairpin RNA folding. At the same time, NusA increases the spectrum of the pausing signals recognized by P7 and allows it to suppress backtracking-stabilized and some elemental pauses, but not the consensus pause. Therefore, the pathways of RNAP pausing appear to be different for different types of pause-inducing signals.
2. Materials and Methods 2.1. Proteins and nucleic acids X. oryzae core RNAP was purified from X. oryzae cells as described previously [29, 30]. The P7 protein of phage Xp10, the σA factor and NusA of X. oryzae were expressed and purified from E. coli [29]. E. coli GreA and GreB were obtained as described previously [29, 40]. DNA and RNA oligonucleotides used for TEC reconstitution were synthesized by DNA Synthesis and Evrogen (Moscow). 2.2. Transcription in vitro Analysis of hairpin-stabilized and consensus pausing was performed using synthetic oligonucleotide scaffolds containing 5′-labeled RNA (Fig. 1A and 2A), as described previously [13, 18, 29, 41]. Reconstituted TECs were incubated with 100 µM UTP, 100 µM CTP and 2 µM GTP (for hisP), or 100 µM CTP and 100 µM GTP (for consP) for increasing time intervals; chase reactions were performed with 1 mM NTPs after the final time point for additional 1 min. The reactions were stopped by the addition of an equal volume of EDTA/urea-containing stopbuffer, RNA products were analyzed by 19% denaturing PAGE and quantified by phosphorimaging using a Typhoon 9500 scanner (GE Healthcare). The efficiency of pausing was calculated as a ratio of the paused transcript to the sum of paused and read-through RNAs, with subtraction of residual pausing in the chase reactions. The data were fit to the single-exponential equation. Analysis of σ-dependent pausing was performed using synthetic oligonucleotide scaffold (Fig. 3A) as described in ref. [40, 42]. Briefly, reconstituted TECs containing 20 nM of 5′labeled RNA, 40 nM of the template DNA strand, 200 nM of the nontemplate DNA strand and 40 nM of X. oryzae core RNAP were supplied with 1 µM σA subunit, 1.5 µM P7 and/or 0.7 µM NusA; GreB was added to 1 µM when indicated. The TECs were incubated with all four NTPs (100 µM each) at 30°C for increasing time intervals, followed by analysis of RNA products by 3
PAGE 19% and phosphorimaging. The relative pause efficiency was calculated as the ratio of the paused RNA products (24/25 nt) in each sample to the amount of paused RNA at the first time point in reactions containing the σA subunit nut lacking other factors. Analysis of oligoU-dependent pausing was performed on a PCR DNA template containing the U-tract from the λ tR2 terminator placed downstream of the λ PR promoter with a 26-mer Cless initially transcribed sequence (Fig. 4A) as described previously [29]. 26-mer TECs containing radiolabeled RNA were obtained by performing transcription with limited substrate set (25 µM ApU, 100 µM ATP, 100 µM GTP with addition of α-[32P]-UTP), followed by the addition of P7 (5 µM), GreA (2 µM), and/or NusA (0.5 µM) and all four NTPs (100 µM ATP, GTP, CTP, 25 µM UTP) at 30 °C.
3. Results 3.1. Distinct effects of P7 and NusA on hairpin-stabilized and elemental pausing at the his pause signal To assess the role of RNA folding in the antipausing activity of P7, we analyzed hairpinstabilized pausing by X. oryzae RNAP using reconstituted TECs, in which RNA hairpin formation could be mimicked by the addition of antisense RNA. The TECs were reconstituted from the core RNAP and synthetic oligonucleotides, based on the sequence of the his pause (hisP) signal from the histidine biosynthesis operon of enterobacteria (Fig. 1A). In the absence of RNA hairpin, this sequence was shown to induce elemental pausing, depending on RNAP interactions with the RNA-DNA hybrid, upstream and downstream forks and downstream DNA duplex [1, 17]. In the presence of RNA hairpin, such complexes recapitulate the properties of paused TECs containing full RNA hairpin obtained during promoter-dependent transcription in vitro [18]. The RNA oligonucleotide used for TEC reconstitution did not contain hairpin, thus allowing to analyze elemental pausing. The hairpin formation was mimicked by the addition of a short antisense RNA oligonucleotide (asRNA) complementary to the upstream part of the RNA transcript (Fig. 1A). We first tested whether P7 could suppress transcriptional pausing in reconstituted complexes. In agreement with its reported effects on hairpin-stabilized pausing [29], P7 decreased pausing in TECs reconstituted in the presence of asRNA, confirming that these complexes can be used to study the antipausing activity of P7: the pause half-life time (t1/2) was decreased 3-fold, from 61.8 ± 2.2 s in the absence of P7 to 20.2 ± 2.7 s in its presence (Fig. 1B and C, filled symbols). As expected, the pause half-life was significantly decreased in the absence of asRNA (t1/2 = 15 ± 1.2 s). Strikingly, in this case, P7 had no effect on RNAP pausing or even slightly increased it (t1/2 = 17.8 ± 1.6 s; Fig. 1B and C, open symbols). Furthermore, in the presence of P7 the pause half-lives were identical independently of the asRNA addition (17.8 ± 1.6 s vs. 20.2 ± 2.7 s, Fig. 1B), suggesting that P7 likely acts by preventing RNA duplex formation in the RNA exit channel of RNAP, in agreement with structural data [25]. We next analyzed the effects of P7 on pausing in the presence of NusA. NusA normally stimulates the recognition of pausing and termination signals by bacterial RNAP but can be recruited by phage-encoded proteins, including P7, to act as a cofactor during antitermination [1, 22, 43]. It was previously demonstrated that NusA stimulates the antipausing activity of P7 and also increases its apparent affinity to RNAP [29]. We showed that X. oryzae NusA indeed increased pausing in reconstituted TECs containing asRNA in the absence of P7 (t1/2 of 84.2 ± 5.0 s). In agreement with published data [18], this increase was modest in comparison with TECs containing longer RNA duplexes or full his RNA hairpin, but significant. In contrast, 4
NusA stimulated the antipauing effect of P7 in comparison with NusA-less complex (t1/2 of 11.3 ± 2.0 s vs. 20.2 ± 2.7 s in the absence of NusA) (Fig. 1D). NusA had no strong effect on elemental pausing in hairpin-less TEC, reconstituted in the absence of asRNA (t1/2 of 21.1 ± 1.9 s in comparison with 15 ± 1.2 s without NusA). However, P7 together with NusA suppressed pausing in the absence of asRNA to the same level as in its presence (t1/2 of 10.2 ± 2.0 s vs. 11.3 ± 2.0 s). Furthermore, the half-life time of elemental pausing (determined in the absence of asRNA) in the presence of both P7 and NusA was significantly (p<0.05) smaller than the half-life time measured in their absence (t1/2 of 10.2 ± 2.0 s vs. 15.0 ± 1.2 s, Fig. 1B and C) demonstrating that both proteins cooperate to suppress elemental pausing at the hisP signal independently of RNA hairpin formation. 3.2. P7 and NusA do not suppress consensus pausing The consensus pause was discovered previously in the whole-genome NET-seq experiments in E. coli, resulting in the identification of a widespread pausing signal, the most conserved features of which are the -1C/T, +1G motif near the RNA 3’-end, and the -11G, -10G dinucleotide at the upstream border of the RNA/DNA hybrid (Fig. 2A) [12-14]. A similar pause sequence was identified in B. subtilis [13]. Further analysis demonstrated that it has a multipartite nature and likely stabilizes the TEC in the half-translocated conformation, similarly to the hisP signal. It was therefore defined as the consensus elemental pause signal (consP), likely underlying most pausing events [17] (see Introduction). We previously showed that, in contrast to hairpinstabilized pausing, P7 could not suppress consensus pausing or even slightly increased its duration [29]. Since the experiments presented in Fig. 1 demonstrated that NusA can assist P7 to suppress elemental pausing, we tested whether it could also help P7 to prevent pausing at the consensus pause site. Analysis of consensus pausing was performed in reconstituted TECs containing the previously established consensus pause sequence (Fig. 2A) [13]. In accordance with published data, P7 did not affect or even slightly increased the pause half-life (t1/2 of 16.6 ± 3.2 s vs. 22.5 ± 4.3 s, Fig. 2B and C; see also Fig. S1 below) [29]. It should be noted that in this case transcription was performed at higher NTP concentrations than in the case of his elemental pause, because consP is a stronger pause signal with a longer half-life time, e.g. [1, 16, 17]. Strikingly, NusA did not have any significant effect on pausing both in the absence and in the presence of P7 (t1/2 of 14 s and 21 s,respectively; Fig. 2B and D). Therefore, the consensus pause is functionally different from the his elemental pause present in the hisP signal, in that it cannot be suppressed by simultaneous action of P7 and NusA. Since P7 and NusA bind near the RNA exit channel of RNAP, we tested whether the synthetic oligoU sequence present in the RNA 5’-end in the consP complexes could affect formation of paused complexes and their sensitivity to P7 and NusA, by analyzing a modified version of the consP scaffold containing the 5’-RNA sequence from the hisP complex (Fig. S1A). It was found that this modification did not strongly change the pause half-life times either in the absence or in the presence of P7 or NusA in comparison with the original scaffold, and P7 together with NusA even increased the pause duration (Fig. S1B). 3.3. P7 and NusA cooperate to suppress backtracking-stabilized pausing TEC backtracking can lead to prolonged RNAP stalling or permanent arrest of transcription [4446]. Backtracked TECs can be reactivated by transcript cleavage factors, GreA in most bacteria, GreA or GreB in E. coli and X. oryzae, or TFIIS in the case of eukaryotic RNAP II. Several 5
previously characterized pause signals were shown to be associated with backtracking, including σ-dependent pauses [40, 47-49], oligoU sequences [29, 31] and ops sites [3, 50], as revealed by their suppression by Gre factors. The consensus paused complex can also readily adop a backtracked conformation sensitive to Gre-induced RNA cleavage, but this does not seem to contribute to the pause half-life [13, 17]. To reveal whether P7 and NusA can affect RNAP backtracking, we tested their action on the formation of backtracking-stabilized pauses of various types by X. oryzae RNAP. The existence of σ-dependent pausing in X. oryzae has never been studied. To reveal whether the σA factor of X. oryzae can induce pausing by its RNAP, we reconstituted TEC on a synthetic template containing the consensus σ-dependent pause signal (TGnTATAAT, where ‘n’ is any nucleotide), which was used previously to analyze σ-dependent pausing by RNAPs from various groups of bacteria [40, 42, 51, 52] (Fig. 3A). The expected pause position was located 35 nucleotides downstream of the 3’-end of the starting RNA transcript, corresponding to the site of pausing previously observed for the σ70- and σA-dependent pauses for E. coli, Thermus aquaticus and Deinococcus radiodurans RNAPs [40, 42, 47, 49, 51]. We found that in the presence of X. oryzae σA transcription was efficiently stalled at the expected pause site (24-26 nt RNAs), with pause half-life exceeding 5 minutes (Fig. 3B and C, compare panels 1 and 2). GreB decreased the efficiency of pausing at positions 24/25 and fully suppressed pausing at position 26 (Fig. 3B and C, panel 6), confirming that the paused complexes adopt a backtracked conformation and can be reactivated by RNA cleavage [40, 42]. P7 or NusA alone had only small effects on σ-dependent pausing (panels 3 and 4). However, when present together, P7 and NusA significantly reduced the efficiency of pausing (to 55-60%, p<0.01) (panel 5). At the same time, P7 plus NusA did not decrease the duration of pausing (little pause escape was observed at the 5 minute reaction point), suggesting that they suppress initial pause formation rather than decrease the pause dwell time. The ops sequence is a pause-inducing signal located in promoter-proximal regions of certain operons in E. coli and other proteobacteria and required for recruitment of an antitermination factor RfaH for efficient transcription of downstream genes. The 12 nt ops signal can be specifically recognized by RNAP and RfaH in the nontemplate DNA strand and induce limited backtracking (Fig. S2A) [3, 50, 53]. Previously, it was shown that P7 can suppress pausing at the ops sequence [39], although the antipausing effect might be milder than for hairpin-dependent pausing and termination signals [29, 39]. We tested whether NusA could enhance the antipausing activity of P7 on an ops-containing template. Interestingly, under conditions of our experiments P7 itself had only small if any effect on ops-dependent pausing by X. oryzae RNAP. At the same, the longevity of ops pause was strongly decreased in the presence of both P7 and NusA (Fig. S2B). OligoU-motifs represent another type of a pause-inducing signal that is associated with TEC backtracking. In the context of intrinsic terminators, the oligoU sequence first induces transient pausing by RNAP, followed by RNA hairpin folding that further destabilizes the TEC and leads to its dissociation [8]. During termination the RNA hairpin formation likely prevents backward RNAP translocation, but isolated oligoU-motifs can promote TEC backtracking, likely due to unstable RNA/DNA hybrid formed in the RNAP active center [29, 31]. In agreement with our previous report [29], X. oryzae RNAP paused at several sites at the oligoU sequence, taken from the λ tR2 terminator and placed downstream of a 26-mer C-less sequence under the control of the λ PR promoter (Fig. 4A; panel 1 in Fig. 4B). RNAP stalling at these sites was suppressed by GreA, confirming the formation of backtracked TECs during transcription (Fig. 4B, panel 3). P7 had no significant effect on pausing, when present either alone or together with GreA (Fig. 4B, compare panels 1, 2 and 3, 4). Thus, P7 by itself cannot prevent RNAP backtracking and 6
does not cooperate with GreA in reactivation of backtracked TECs. At the same time, P7 together with NusA suppressed oligoU-dependent pausing (Fig. 4B, panel 6), in accordance with published data [29], while NusA alone stimulated pausing (panel 5). The above results demonstrated that P7 and NusA can suppress different types of backtracking-stabilized pauses. To reveal whether P7 and NusA might also directly reactivate backtracked TECs, we analyzed their effects on RNAP activity in reconstituted TEC containing a mismatched nucleotide at the RNA 3’-end (Fig. S3A). The mismatch promotes one-nucleotide backtracking of the TEC with subsequent cleavage of the 3’-dinucleotide from the RNA transcript, resulting from the intrinsic RNA cleavage activity of RNAP [54, 55]. To avoid premature RNA cleavage during TEC assembly, the complex was obtained in the absence of Mg2+ ions. Incubation of the reconstituted TEC with NTP substrates and Mg2+ led to both extension and cleavage of the starting RNA transcript (Fig. S3B). Because of the presence of mismatch, the kinetics of RNA extension in this complex was much slower than in the case of fully-matched complexes (e.g. [54]). NusA and P7 did not have an inhibitory effect on RNA cleavage or a stimulatory effect on the extension of the mismatched RNA by X. oryzae RNAP (Fig. S5B and S5C). In fact, the kinetics of RNA extension in the presence of both P7 and NusA was even slower than in the absence of factors (Fig. S5C). This result suggested that P7 and NusA cannot reactivate preformed backtracked TECs.
4. Discussion 4.1. Antitermination and antipausing activities of P7 Transcription antitermination is an essential process in the regulation of bacterial and bacteriophage gene expression [22]. Previous studies identified several cellular and phageencoded regulatory proteins that act as transcription antiterminators, but for most of them the molecular mechanisms of action have remained only partially understood [24-30]. Here, we analyzed the antipausing activity of the P7 antiterminator protein encoded by phage Xp10 of X. oryzae and revealed that it has distinct effects on various types of transcriptional pauses. Thus, this small regulatory protein can discriminate between different functional states of the TEC, likely underlying its specific effects on transcription termination. Previous studies suggested that various types of transcriptional pauses, including those studied in this work, are formed through the same intermediate, the elemental pause [1, 16, 17]. The two best studied elemental pause signals, hisP (without RNA hairpin) and consP, have several common nucleotides at key positions (-10, -1 and +1; Fig. 1A and Fig. 2A), suggesting that both complexes may be functionally similar. Their analysis demonstrated that the elemental paused complex can isomerize between the preferable half-translocated and less-populated pretranslocated or backtracked conformations. Changes in various parts of the pause signal may possibly affect the equilibrium between these states and change the functional properties of the paused complex [1, 17]. During hairpin-stabilized pausing, the RNA hairpin formation stabilizes the half-translocated TEC conformation and induces ‘swiveling’ of several domains of the RNAP β′ subunit, including clamp, shelf, jaw and the SI3 insertion in the trigger loop in the active center, around an axis roughly coinciding with the bridge helix of RNAP (Fig. 5A) [4, 16]. The swiveled conformation inhibits trigger loop folding by changing the position of the SI3 insertion, further inhibiting nucleotide addition. The universal bacterial factor NusA stimulates hairpin-stabilized, but not elemental, pausing by binding near the RNA exit channel and directly interacting with the flap domain of RNAP and nascent RNA, thus promoting hairpin formation (Fig. 5A) [4, 16, 43]. 7
We showed that P7 specifically suppresses hairpin-stabilized pausing by bacterial RNAP, likely by preventing RNA folding in the RNA exit channel of RNAP. Indeed, the duration of the his pause in the presence of P7 was decreased to the level of the hairpin-less elemental pause with the same sequence, which by itself was not affected by P7. These observations are supported by the recently published structure of TEC of X. oryzae RNAP with bound P7 [25]. In this structure, the RNA exit channel between the β flap domain and the main RNAP body is constricted by P7 and is too narrow for RNA hairpin formation (Fig. 5B). We showed that the activity of P7 at the hisP signal and its affinity to the TEC are enhanced by NusA ([29] and this work). As revealed in the TEC structure, P7 binding is directly stimulated by NusA that interacts with P7 at the RNA exit channel (Fig. 5C) [25]. Together, P7 and NusA may stabilize the closed conformation of the TEC and likely prevent RNAP swiveling, thus explaining the stimulatory effects of NusA on the antipausing activity of P7. In contrast to hairpin-stabilized pausing and intrinsic termination, P7 does not significantly affect other types of analyzed pauses, including his elemental and consensus elemental pauses, as well as backtracking-associated oligoU-dependent and σ-dependent pauses. This suggests that P7 likely cannot prevent conformational changes of the TEC associated with formation of these pauses. In comparison, NusA is also unable to regulate transcriptional pauses which are not associated with RNA hairpin formation in the RNA exit channel [3, 18, 43]. Strikingly, however, P7 in the presence of NusA can suppress the his elemental pause and various types of backtracking-stabilized pauses, including σ-dependent and ops pauses which also contain the elemental pause motif, in particular, G at position +1 (Fig. 3, Fig. S2) [56, 57]. Thus, when P7 and NusA are simultaneously bound to these TECs they might counteract elemental pausing associated with isomerization of the TEC into the inactive half-translocated conformation. P7 was shown to promote forward translocation of the TEC [39]; while this activity is apparently insufficient for suppression of elemental and backtracking-stabilized pausing by P7 alone, it may be further enhanced by NusA. Furthermore, since both factors bind at the upstream part of the swiveling module of RNAP, they might physically prevent its rotation that could transiently occur during elemental pausing (Fig. 5C) [1, 16, 17]. At the same time, these factors cannot apparently reactivate already backtracked complexes, as demonstrated by the absence of their effect on the longevity of σ-dependent pauses (Fig. 3) or on RNAP activity in backtracked mismatched complexes (Fig. S3). Recent structural analysis of a backtracked TEC revealed that it mostly adopts a swiveled conformation, similar to the elemental paused complex [4, 16, 58]. However, the location of the RNA 3’-end in the secondary channel as a result of backward TEC translocation likely makes impossible direct activation of such complexes by P7 and NusA, even if these factors could reverse RNAP swiveling. Intriguingly, the consensus elemental pause, which acts as a universal pause signal in various bacteria and even eukaryotes [13], is resistant to the action of P7 and NusA, in contrast to the his elemental pause and backtracking-stabilized pauses containing the elemental pause motifs. This result confirms that the consensus pause signal has a multipartite nature and the weekly conserved positions in this motif, identified in refs. [12, 13] (shown in plain letters in Fig. 2A), contribute to its recognition by RNAP. As a result, the TEC formed at the consensus pause site may be either more strongly stabilized in the same conformation as in the case of the his elemental pause (due to stronger RNAP interactions with the consensus sequence), or adopt another conformation different from the his elemental pause, thus making it insensitive to P7 and NusA. Therefore, although both complexes were proposed to fluctuate between different translocation states [12, 16, 17, 59], they may have different preferable conformations resulting in their different sensitivity to the antipausing action of P7 and NusA.
8
4.2. Antipausing activities of various antitermination factors Analysis of bacteriophage antiterminator proteins reveals that, in addition to antitermination, all these factors can suppress various types of transcriptional pauses. Most studied antiterminators, including P7 of phage Xp10, N and Q of λ, gp39 of P23-45, and put RNA of HK022, bind at different sites around the RNA exit channel of RNAP (Fig. 5B,C). Accordingly, N [27], Q [26] and P7 (this work and [25]) were shown to prevent RNA folding in the RNA exit channel and to protect nascent RNA from the action of pro-termination factors. Recent structural analysis of these three proteins, N, Q and P7, confirmed that they interfere with RNA hairpin formation, thus explaining their effects on intrinsic transcription termination and hairpin-stabilized pausing [24, 25, 27]. At the same time, these antiterminator proteins rely on different sets of RNA/RNAP interactions for their antipausing/antitermination activities. Both N and P7 recruit NusA to rearrange the RNA exit channel; however, while the former protein destabilizes RNA hairpin formation by moving NusA and the flap domain of RNAP away from nascent RNA, the latter one constricts the RNA exit channel and likely redirects the transcript through an alternative way out of RNAP [25, 27]. In contrast, analysis of the Q protein from the lambdoid phage 21 demonstrated that it forms a narrow ring on nascent RNA at the exit from RNAP, thus effectively shielding it from secondary structure formation or termination factor binding [24, 60]. It remains to be known whether NusA can stimulate the antipausing activity of Q21 and its loading onto RNA, similarly to previously studied proteins Qλ and Q82 [26, 61, 62]. Different groups of antitermination factors may target common intermediates in the pausing pathway even if they bind at different sites of RNAP. P7 and other bacteriophage proteins mentioned above prevent RNA folding by changing the conformation of the RNA exit channel and restricting RNAP opening, thus stabilizing the active state of the TEC. The bacterial antiterminator RfaH acts in a similar way, while binding at the upstream part of the DNA binding cleft of RNAP [5]. RfaH was also shown to prevent RNAP swiveling [5], similarly to the proposed cooperative action of P7 and NusA. Further analysis of the antipausing and antitermination activities of small phage-encoded protein regulators may provide deeper insights into the intricate conformational mobility of the TEC, help to discover new antipausing mechanisms and develop robust synthetic switches for the regulation of gene expression in bacteria.
Authors’ contributions AK and DE conceived the study and designed experiments, MP and DE performed experiments and analyzed data. AK wrote the manuscript. All authors read and approved the manuscript.
Conflict of Interest The authors declare that there is no conflict of interest.
Acknowledgements We thank Dr. Ivan Petushkov for useful discussions, I. Artsimovitch for plasmids.
Funding This work was supported in part by the Russian Science Foundation (grant 17-14-01393 to AK, analysis of transcription antitermination) and Russian Foundation for Basic Research (grant 1834-20095 to DE, analysis of σ-dependent pausing).
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Figure legends Figure 1. Effects of P7 and NusA on hairpin-stabilized and elemental pausing at the his pause site. (A) Scheme of the nucleic acid scaffold based on the hisP sequence and used for TEC assembly; DNA is black RNA is red. The TECs were reconstituted from synthetic oligonucleotides either with or without addition of asRNA, followed by addition of P7 and/or NusA and NTPs (CTP, UTP, GTP), resulting in the extension of the starting 17 nt RNA by four nucleotides. The 19 nt paused RNA is indicated with an arrow. Nucleotides corresponding to the consensus pause sequence are shown in bold (see Fig. 2A) [12, 13]. Nucleotide positions relative to the pause sites are numbered above the scheme. (B) Analysis of the kinetics of RNA extension in the absence of NusA. Chase reactions (‘C’) were performed with the addition of high concentrations of NTPs after the final time point. Positions of the starting 17 nt, paused 19 nt and readthrough 21 nt RNAs are indicated. Note that a fraction of the starting RNA was not extended likely because it was not bound by RNAP during TEC reconstitution. (C) Kinetics of pausing. At each time point, the efficiency of pausing was quantified as the ratio of paused to the sum of paused and readthrough RNAs, with subtraction of paused RNAs in the chase reaction (‘C’). The pause half-life times are shown on the right (means and standard deviations from three independent experiments). Figure 2. Effects of P7 and NusA on pausing at the consP pause site. (A) Scheme of the nucleic acid scaffold used for TEC assembly. The consensus pause sequence (‘Cons’) is shown above the scheme; the most conserved positions are bold [12, 13]; nucleotides are numbered relative to the pause position. The TECs were reconstituted from synthetic oligonucleotides, followed by the addition of P7 and/or NusA and NTPs (GTP, CTP), resulting in the extension of the starting 15 nt RNA by seven nucleotides. The 17 nt paused RNA is indicated with an arrow. (B) Analysis of RNA extension in the absence and in the presence of P7 and/or NusA. Positions of the starting 15 nt, paused 17 nt and readthrough 22 nt RNAs are indicated. The unextended fraction of 15 nt RNA corresponds to RNA that was not bound by RNAP during TEC reconstitution. (The samples in NusA-containing reactions were separated on the same gel, and a part of the gel was cut off to remove empty lanes). (C) Kinetics of pausing. The pause half-life times are shown on the right (means and standard deviations from three independent experiments). Figure 3. Analysis of σ-dependent pausing by X. oryzae RNAP. (A) Scheme of the nucleic acid scaffold used for TEC assembly. The TECs were reconstituted from synthetic oligonucleotides, followed by addition of the σA subunit, P7 and/or NusA and all four NTPs. Positions of the -10-like element, starting 20 nt and paused 25 nt RNAs are indicated. Nucleotides corresponding to the consensus pause sequence are shown in bold (positions -11, -1 and +1; see Fig. 2A) (B) Analysis of σ pause formation in the absence and in the presence of P7, NusA and GreA. Positions of the starting 20 nt, paused 25 nt and run-off (RO) RNAs are indicated. The panel numbers are indicated under the gel. (C) Quantification of pause efficiencies. For each reaction point, the efficiency of pausing at positions 24/25 is shown relative to pausing measured at the 0.5 min time point in the presence of σA and in the absence of other factors (means and standard deviations from three independent experiments). Figure 4. Analysis of oligoU-dependent pausing by X. oryzae RNAP. (A) The initially transcribed sequence of the oligoU template. The +1 and +26 positions are indicated, the oligoUmotif from the λ tR2 terminator is shown in pink, the 31-37 nt pausing region is shown with blue line. (B) PAGE-analysis of RNA products synthesized on the oligoU template. Positions of the starting 26-mer RNA, 31-37 nt paused RNA products and full-length run-off (RO) RNA are indicated. The chase reactions (‘C’, last lane in each panel) were performed with high 14
concentrations of NTPs (1 mM each) added after the 10’ time point. The panel numbers are indicated under gel. Figure 5. Structural models of paused and P7-bound TECs. (A) Hairpin-containing E. coli TEC with bound NusA at the hisP pause signal. The direction of RNAP swiveling in the paused complex in comparison with active TEC is shown with a blue arrow. (B) TEC of X. oryzae RNAP with bound P7 (shown as a light blue surface). (C) TEC of X. oryzae RNAP with bound P7 and NusA; the narrow RNA exit channel between the Zn-binding domain (ZBD), P7 and β flap is shown with an arrow. The RNA transcript is yellow, the flap domain in the RNAP β subunit is red, the ZBD in the β’ subunit is brownish, NusA is light green. The N-terminus of the β’ subunit involved in P7 binding is black. The PDB accession numbers for each structure are indicated.
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Highlights: • • • •
Small antiterminator protein P7 of phage Xp10 modulates pausing by RNA polymerase P7 prevents RNA duplex formation during hairpin-stabilized pausing P7 cooperates with NusA to suppress elemental and backtracking-stabilized pauses The consensus elemental pause is resistant to the action of P7 and NusA