Function of the Bacillus subtilis elongation factor NusG in hairpin-dependent RNA polymerase pausing in the trp leader

Alexander V. Yakhnin, Helen Yakhnin, and Paul Babitzke*

Department of Biochemistry and , Pennsylvania State University, University Park, PA 16802

Communicated by Charles Yanofsky, Stanford University, Stanford, CA, September 5, 2008 (received for review June 19, 2008) NusA and NusG are transcription elongation factors that bind to transcript participate in the attenuation mechanism. An antiter- RNA polymerase (RNAP) after ␴ subunit release. minator structure (AT) can form just upstream from an intrinsic NusA (NusAEc) stimulates and RNAPEc pausing, hairpin (T). Because these two structures overlap by whereas NusGEc promotes Rho-dependent termination and pause 4 nucleotides (nt), their formation is mutually exclusive. Tryp- escape. Both Nus factors also participate in the formation of RNAPEc tophan-activated TRAP binds to the nascent trp leader tran- antitermination complexes. We showed that Bacillus subtilis NusA script and blocks formation of the AT. Thus, TRAP promotes (NusABs) stimulates intrinsic termination and RNAPBs pausing at termination by favoring formation of the T (Fig. 1). When U107 and U144 in the trpEDCFBA leader. Pausing at U107 is limiting, TRAP does not bind to the trp transcript, and U144 participates in the transcription attenuation and trans- thus the AT forms, and the operon is expressed (7). Interaction lational control mechanisms, respectively, presumably by provid- of TRAP with a stem–loop that forms at the 5Ј end of the trp ing additional time for trp RNA-binding attenuation protein (TRAP) transcript (5ЈSL) increases the efficiency of termination (8). In to bind to the nascent trp leader transcript. Here, we show that the control mechanism, binding of TRAP to the same NusGBs causes modest pause stimulation at U107 and dramatic triplet repeats in trp operon readthrough transcripts promotes pause stimulation at U144. NusABs and NusGBs act synergistically to formation of a structure that sequesters the trpE Shine–

increase the U107 and U144 pause half-. NusGBs-stimulated Dalgarno (SD) sequence (Fig. 1). This structure inhibits TrpE BIOCHEMISTRY pausing at U144 requires RNAPBs, whereas NusABs stimulates synthesis by preventing binding. In the absence of pausing of RNAPBs and RNAPEc at the U144 and E. coli his pause bound TRAP, an alternative RNA structure forms in which the sites. Although NusGEc does not stimulate pausing at U144, it trpE SD sequence is available for ribosome binding (7). Because competes with NusGBs-stimulated pausing, suggesting that both translation of trpE and trpD is coupled, formation of the trpE SD proteins bind to the same surface of RNAPBs. Inactivation of nusG sequestering hairpin also regulates TrpD synthesis. Inhibition of results in the loss of RNAP pausing at U144 in vivo and elevated trp trpE translation also causes transcriptional polarity, which re- operon expression, whereas plasmid-encoded NusG complements duces expression of the downstream genes. However, inhibition the mutant defects. Overexpression of nusG reduces trp operon of trpE translation does not affect the rate of mRNA decay (7). expression to a larger extent than overexpression of nusA. We found that B. subtilis NusA (NusABs) increases the ter- mination efficiency in the trp leader and stimulates B. subtilis transcription attenuation ͉ translational control ͉ NusA RNAP (RNAPBs) pausing at two hairpin-dependent pause sites (U107 and U144) (9). U107 is the nucleotide just preceding the usA and NusG from Escherichia coli (NusAEc and NusGEc, critical overlap between the AT and T structures (Fig. 1). Nrespectively) function as general transcription elongation Although pausing at U107 presumably provides additional time factors. Binding of these two proteins to E. coli RNA polymerase for TRAP to bind and promote termination, it has not been (RNAPEc) typically occurs after escape and dissocia- possible to establish that pausing at this position occurs in vivo tion of the RNAP ␴ subunit (1). Both NusAEc and NusGEc (9, 10). However, pausing at U144 was shown to occur in vivo, increase the RNAPEc elongation rate during transcription of and pausing at this site reduces expression of the trp operon (10). rRNA and certain protein-coding genes (2). nusA and Pausing at U144 participates in the trpE translation control nusG are normally essential E. coli genes; however, inactivation mechanism, apparently by providing a second opportunity for of nusG was possible after deletion of the rac prophage from the TRAP to bind to the nascent trp leader transcript (10). Thus, E. coli chromosome (3). NusAEc and NusGEc have strikingly RNAPBs pausing is capable of synchronizing the RNAP position different activities during transcription in vitro. NusAEc stimu- with TRAP binding and thereby influences RNA folding. Here, lates intrinsic transcription termination and RNAPEc pausing at we demonstrate that B. subtilis NusG (NusGBs) stimulates paus- the hairpin-dependent his pause site but does not affect hairpin- ing at both trp leader pause sites, although NusGBs exerts its independent pausing at an ops pause site (4, 5). In contrast, greatest effect on pausing at U144. We further show that NusGBs NusGEc promotes RNAPEc pause escape from the ops site but has a stronger stimulatory effect on pausing than NusABs at does not affect pausing at the his site (5). NusGEc also serves as U144 and that these two proteins act synergistically to stimulate a bridge between Rho and RNAP in facilitating transcript pausing at both pause sites. We also found that NusGBs is release (6). required for pausing at U144 in vivo and that this protein is The Bacillus subtilis trp RNA-binding attenuation protein essential for proper regulation of trp operon expression. (TRAP) negatively regulates expression of the trp operon by transcription attenuation and translational control mechanisms (7 and references therein). Binding of tryptophan induces a Author contributions: A.V.Y., H.Y., and P.B. designed research; A.V.Y. and H.Y. performed structural reordering of 11 KKR motifs in the 11-subunit TRAP research; A.V.Y., H.Y., and P.B. analyzed data; and A.V.Y. and P.B. wrote the paper. complex, allowing each motif to interact with one of 11 triplet The authors declare no conflict of interest. repeats in the leader transcript, thereby wrapping the RNA *To whom correspondence should be addressed. E-mail: [email protected]. around the TRAP perimeter (Fig. 1). Another protein called This article contains supporting information online at www.pnas.org/cgi/content/full/ anti-TRAP competes with mRNA for the TRAP RNA-binding 0808842105/DCSupplemental. surface (7). Several RNA structures that form in the trp leader © 2008 by The National Academy of Sciences of the USA

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0808842105 PNAS ͉ October 21, 2008 ͉ vol. 105 ͉ no. 42 ͉ 16131–16136 Downloaded by guest on September 30, 2021 G U A U G G C

5’ Stem-loop (5’SL) 80.A U Antiterminator (AT) 60 . GUAGCAG G A A U G G GA AG A U A G.20 U A U G Terminator (T) U G A A A U G A 120 C G A G . G Limiting tryptophan AG C U A U A CU

U A U U C G Little active TRAP 100 G U G U

A U . A A

A U A U G . UA

U

G A A U U U G

U A G A TRAP 80 UA Excess tryptophan G G

A U G U U

A A

A A C U U G C

G A G C U U G

G

A A G A U Active TRAP A A

GA A U U G G

U A U A U107 Pause U A U

C

G G

U A

U A G C A A U

G G G

40 120 G

U

A

A

. A A U G

G C . G 141140

G C G G

A A U U A A

AG C UAA UAG UAA GAG UU GAGU AU GAG AA UAG. C UGAAGAAAA ... . C G 40 C 60 100 C G A . C G Transcription Transcription 120. UUAUGUUUAUUCUA U UUAUUUGU termination readthrough CU Pause G U A U hairpin A U G C Transcription fails A U A U to terminate A U A U 140. U144 Pause C G UUG AU U AUU C G UA U C C G U U

. U UA UU A 100 U U . 60 GUAGCAG G C A UC A G A A U G GA SD-sequestering A UA G C C G A U U U U U G A U A A A A hairpin UUUU A 120 G C G . G U A U C U A CU

Limiting tryptophan C G U Pause AC U G C G U G U Excess tryptophan G U

A A

U A Little active TRAP A . UA hairpin U A

G C U G

TRAP G 80 UA Active TRAP U A G G

UU AU U C G A A

G C U CG SD

G G G

A U G A U 180C G

A A

UU U U .U A

G G

UG C U A U anti-SD C C G

G G

U A U

A U A A U U144 Pause

G G

G G

U

A

A U

A A U

G G

G G C G

A A A U U U A A

U A . C G C G

40 C A U 100 C G 160 U A A U A . C G . C G Met anti-anti-SDG C anti-SD UUAUGUUUAUUCU AUUUAUUUGUUAUAUAGUAUUUUAUCCUCUCAUGCCAUCUU AGGUAAC ... A U.180 . G C 200 A C Efficient CG U Translation inhibition A U translation U G 200 40. G C . Met GAU UAA G GA UU AG GU AU AG GAAGGAU . CACU AGUGAGGAA AG G GUAA ... 60 SD

Fig. 1. Models of B. subtilis trp operon regulation. (Upper) Transcription attenuation model. During transcription RNAP pauses at U107. Under limiting tryptophan conditions, TRAP does not bind to the RNA. Once RNAP resumes transcription, the AT forms, resulting in transcription readthrough. Under excess tryptophan conditions, TRAP binds to the 5ЈSL and the (G/U)AG repeats. Bound TRAP releases paused RNAP and prevents AT formation. Thus, formation of the T causes transcription to terminate at G140 or U141. Because termination is never 100% efficient, a fraction of RNAP molecules will not terminate despite the presence of bound TRAP. (Lower) trpE translational control model. During transcription of readthrough transcripts, RNAP pauses at U144. Under limiting tryptophan conditions, TRAP does not bind to the RNA. Once RNAP resumes transcription, the RNA adopts a structure such that the trpE SD sequence is available for ribosome binding. Under excess tryptophan conditions, TRAP binds to the paused transcript. Once RNAP resumes transcription, the trpE SD-sequestering hairpin forms and inhibits translation of trpE. The same structure functions as the terminator and U144 pause hairpins. The 5ЈSL is shown only in the Upper Left drawing.

Results 1. We also found that NusAEc stimulated pausing of RNAPBs at

NusGBs Stimulates RNAPBs Pausing at the U107 and U144 trp Pause U144 to the same extent as NusABs, whereas NusGEc did not Sites. RNA species corresponding to pause complexes initially affect pausing of RNAPBs (Table 1, rows 5 and 6). increase in abundance and subsequently chase to longer tran- We found that substitution of U144 with A, C, or G reduced scripts. Thus, paused species are readily distinguished from the pause half- when ATP was limiting (10). In the presence terminated transcripts that cannot be extended. Previous in vitro of nonlimiting ATP, pausing was not observed with the U144A mutant in the absence of Nus factors (basal pausing) or in the transcription studies demonstrated that NusABs stimulates paus- ing at U107 and U144 in the B. subtilis trp leader (9, 10). To presence of only NusABs. However, pausing was observed with explore further the mechanism of pausing, experiments were NusGBs alone and in combination with NusABs, resulting in pause half-lives of 32 and 120 s, respectively (Table 1, rows 7–10). performed to determine whether NusG affected pausing at Bs Thus, the U144A substitution results in a 13-fold reduction in the these two sites. Because incorporation of ATP is required for NusG -stimulated pause half-life (Table 1, rows 3 and 9). pause escape from both pause sites (Fig. 1), a limiting concen- Bs ␮ Despite the absence of pausing with NusABs alone, the synergy tration of ATP (10 M) was used in single-round transcription of the Nus factor combination was retained with the U144A assays to increase the dwell time of RNAPBs at U107 and U144. mutant; the addition of NusABs resulted in a 3- to 4-fold increase NusABs and NusGBs individually increased the U107 pause in the NusG -stimulated pause half-life in the wild type (WT) Ϸ Bs half-life 5-fold and acted synergistically when added together, and mutant leaders (Table 1, compare rows 1–4 with 7–10). resulting in a combined 23-fold increase in the pause half-life We next tested the influence of NusA and NusG from B. (Fig. 2A). Because the paused transcription complex (PTC) at subtilis and E. coli, as well as some heterologous Nus factor A U144 was so stable with limiting ATP (Fig. 2 ), pausing combinations, on RNAPEc pausing at the well characterized experiments were also performed with nonlimiting ATP (150 hairpin-dependent his pause site of E. coli (5, 11–13). Because ␮ M). At the higher ATP concentration, NusABs and NusGBs incorporation of GTP is required for pause escape, a limiting increased the U144 pause half-life Ϸ4-fold and Ϸ100-fold, concentration of GTP was used to increase the dwell time at this respectively (Fig. 2B and Table 1, rows 1–3). Again, NusABs and pause site. As was reported in ref. 5, NusAEc stimulated pausing NusGBs acted synergistically, resulting in a combined 300-fold at the his pause site, whereas NusGEc did not stimulate pausing Ϸ increase in the pause half-life ( 22 min) (Fig. 2B and Table 1, alone or in combination with NusAEc (Table 1, rows 11–14). The row 4). Note that the pause half-life values for the gel shown in effect of NusABs and NusGBs on RNAPEc pausing was similar to Fig. 2B differ from the average half-life values reported in Table the effect of the E. coli Nus factors (Fig. 2C and Table 1, rows

16132 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0808842105 Yakhnin et al. Downloaded by guest on September 30, 2021 Table 1. Modulation of RNAPBs pausing at U144 in vitro by NusA and NusG from B. subtilis and E. coli Row Pause site RNAP Nus* Half-life, s

1 U144 Bs 4.3 Ϯ 0.8

2 U144 Bs ABs 17 Ϯ 3 3 U144 Bs GBs 420 Ϯ 150 4 U144 Bs ABs ϩ GBs 1,300 Ϯ 510 5 U144 Bs AEc 16 Ϯ 1 6 U144 Bs GEc 3.3 Ϯ 0.9 7 U144A Bs No pausing

8 U144A Bs ABs No pausing 9 U144A Bs GBs 32 Ϯ 1 10 U144A Bs ABs ϩ GBs 120 Ϯ 11 11 his Ec 38 Ϯ 3

12 his Ec AEc 190 Ϯ 18 13 his Ec GEc 27 Ϯ 6 14 his Ec AEc ϩ GEc 130 Ϯ 21 15 his Ec ABs 160 Ϯ 14 16 his Ec GBs 34 Ϯ 2 17 his Ec ABs ϩ GBs 130 Ϯ 33 18 his Ec AEc ϩ GBs 160 Ϯ 19 19 his Ec ABs ϩ GEc 92 Ϯ 38 Fig. 2. NusABs- and NusGBs-stimulated pausing at the trp U107, trp U144, and his pause sites, and competition of NusGBs with NusGEc at the trp U144 pause Transcription reactions with WT (U144) and mutant (U144A) B. subtilis trp site. Single-round in vitro transcription reactions were performed with the leader templates and RNAPBs (Bs) were carried out at 23°C and contained a 150 indicated Nus factors and stopped at the times shown above each lane. Chase ␮M concentration of each NTP. Reactions with E. coli (his) templates and reactions (Ch) were extended for an additional 10 min at 37°C in the presence RNAPEc (Ec) were carried out at 37°C and contained 10 ␮M GTP and a 150 ␮M

of each NTP (500 ␮M). Pause half-lives (T1/2) from these representative gels are concentration of the other three NTPs. Average pause half-life values are BIOCHEMISTRY shown. (A) Transcription of the trp leader by RNAPBs was performed at 23°C in reported Ϯ SD. the presence of 10 ␮M ATP (limiting) and 150 ␮M concentrations of the other *ABs,AEc,GBs, and GEc are NusA and NusG proteins of B. subtilis (Bs) and E. coli (Ec). three NTPs. Positions of the U107 paused, U144 paused, and runoff (RO) transcripts are shown. The 167-nt band is a pausing artifact caused by the close proximity of this base to the end of the DNA template. Because position 143 shown), whereas only NusA-stimulated pausing was observed at is an A residue, the 142-nt terminated transcript is an artifact of limiting ATP. the U144 pause site (Table S1, rows 13–16). Taken together, our (B) Transcription of the trp leader by RNAPBs was performed at 23°C in the results suggest that NusA proteins are general pause-stimulating ␮ presence of a 150 M concentration of each NTP (nonlimiting). (C) Transcrip- factors and are interchangeable between different RNAPs and tion of the his leader by RNAPEc was performed at 37°C in the presence of 10 ␮M GTP (limiting) and a 150 ␮M concentration of the other three NTPs. hairpin-dependent pause sites of different species, whereas Positions of the his pause and runoff (RO) transcripts are shown. (D) Tran- NusG-stimulated pausing is specific and only occurs when all of scription of the trp leader by RNAPBs was performed at 23°C with a 150 ␮M the components (NusG, RNAP, and pause site) are derived from concentration of each NTP and 0.25 ␮M NusGBs Ϯ 1 ␮M NusGEc. Pause effi- B. subtilis. ciencies (Eff) are shown.

NusGEc Competes with NusGBs-Stimulated Pausing of RNAPBs at the U144 Pause Site. Although NusGBs and NusGEc are 44% identical 11 and 15–17). Furthermore, the pause half-lives of the NusAEc (Fig. S3), NusGEc did not stimulate pausing of RNAPBs at U144 ϩ NusGBs and NusABs ϩ NusGEc combinations were similar to (Table 1, row 6), suggesting either that NusGEc is incapable of the half-lives of the respective NusA proteins alone (Table 1, binding to RNAPBs or that once bound, NusGEc is unable to rows 12, 15, 18, and 19). respond to the pause signal. Hence, we tested whether NusGEc It was shown that RNAP does not pause at the E. coli his Bs could compete with NusGBs-stimulated pausing by simulta- pause site when transcription was performed at 37°C with neously adding both NusG proteins. The NusGBs-stimulated limiting GTP (11). When the temperature was reduced to 23°C pause efficiency dropped Ϸ8-fold when a 4-fold molar excess of and GTP was limiting, a short-lived RNAPBs pause was observed NusGEc was included in the reaction; however, the half-life of the at the same site recognized by RNAPEc; however, because RNAPBs molecules that did pause was only reduced 30% (Fig. several additional pauses were observed with RNAPBs, pausing 2D). The comparable half-life suggested that NusGEc could not of RNAPBs at the his pause site might be nonspecific [supporting displace NusGBs from RNAPBs. This assumption was confirmed information (SI) Fig. S1]. With the exception of a modest by the absence of competition when NusGBs was added to the stimulation by NusABs, Nus proteins from either organism did reaction before the addition of an 8-fold molar excess of NusGEc not stimulate pausing of RNAPBs; however, NusGEc reversed the (Fig. S4). However, the finding that NusGEc competed with pause-stimulating activity of NusABs (Table S1, rows 1–7). NusGBs when the two proteins were added simultaneously Pausing of RNAPEc at the B. subtilis U144 pause site was also suggests that both NusG factors bind to the same surface of examined under a variety of conditions. RNAPEc paused at U144 RNAPBs but that NusGEc does not respond to the U144 pause when transcription was performed at 37°C with limiting ATP. signal. Pausing was stimulated Ϸ15-fold by both NusAEc and NusABs, whereas NusGEc and NusGBs had little if any effect on pausing NusGBs Alters the Accessibility of the Nontemplate DNA (ntDNA) (Fig. S2 and Table S1, rows 8–12). This effect was qualitatively Strand at the U144 Pause Site. Elongating RNAP maintains a similar to the effect of Nus factors on RNAPEc pausing at the his transcription bubble of Ϸ12 nt in which the template DNA and pause site (Table 1, rows 11–13, 15 and 16). Transcription with ntDNA strands are separated. Although Ϸ8 nt of the template RNAPEc at 23°C and nonlimiting NTPs resulted in appreciable DNA strand is sequestered in the RNA–DNA hybrid, the pausing with or without Nus factors at the his pause site (data not ntDNA strand remains single-stranded (1). The unpaired T

Yakhnin et al. PNAS ͉ October 21, 2008 ͉ vol. 105 ͉ no. 42 ͉ 16133 Downloaded by guest on September 30, 2021 Table 2. Effect of nusG and nusA on expression of a trpE؅-؅lacZ fusion ␤-Galactosidase activity, Miller units

Row Genotype ϪTrp ϩTrp ϪTrp/ϩTrp

1WT 33Ϯ 10 0.16 Ϯ 0.02 206 2 ⌬nusG 67 Ϯ 17 0.65 Ϯ 0.02 103 3 ⌬mtrB 310 Ϯ 48 320 Ϯ 16 1 4 ⌬nusG ⌬mtrB 180 Ϯ 8 170 Ϯ 36 1 5 WT/pNusG 12 Ϯ 2ND 6 WT/pNusG (Xyl) 3 Ϯ 0.4 ND 7 WT/pNusA 10 Ϯ 2ND 8 WT/pNusA (Xyl) 5.8 Ϯ 0.9 ND 9 WT/pVector 10 Ϯ 3ND 10 WT/pVector (Xyl) 9 Ϯ 3ND

Cultures were grown at 37°C. Cells without plasmids (top 4 rows) were grown with glycerol and harvested at Klett 75 (midexponential phase). Cells with plasmids (bottom 6 rows) were grown with arabinose and were either not induced or induced with xylose (Xyl) for 4 h and harvested. ␤-Galactosidase activity is reported in Miller units Ϯ SD. ND, not determined.

Fig. 3. Effect of NusGBs and NusABs on footprints of paused transcription bubbles in vitro. Single-round transcription was performed at 23°C with RNAP and the indicated Nus factors with limiting ATP. The DNA template Bs causes a structural rearrangement of the PTC and that NusA was 5Ј end-labeled on the ntDNA strand. Reactions were treated with KMnO4 Bs at the times shown above each lane. Chase reactions (Ch) were extended for increases the rate of this conformational change. an additional 10 min at 37°C in the presence of a 500 ␮M concentration of each NTP before KMnO4 treatment. Positions of the trp pause sites (T107 and T144) NusGBs-Stimulated Pausing Regulates Expression of the trp Operon. and other T residues are marked. KMnO -treated and piperidine-cleaved 4 Because NusGBs causes a dramatic stabilization of the U144 PTC fragments are 1 base shorter than the corresponding base in the DNA se- in vitro,anusG knockout strain was generated to test whether the quence. The relative band intensity of the T residues (133–146) for several Ј Ј lanes is shown below the gel. absence of NusG would affect expression of a trpE - lacZ translational fusion. TRAP-dependent regulation of this fusion in response to tryptophan (ϪTrp/ϩTrp) was Ϸ200-fold (Table 2, residues in the ntDNA strand are subject to oxidation by row 1). Disruption of nusG increased expression of the fusion KMnO4. Because paused RNAP forms a stationary transcription 2-fold or 4-fold when cells were grown in the absence or presence bubble, the T residues are hypersensitive to permanganate of tryptophan, respectively (Table 2, row 2). As expected, oxidation, giving rise to a signature footprint pattern (10). regulation in response to tryptophan was lost in the absence of KMnO4 footprinting was performed in the presence of limit- TRAP (⌬mtrB) (Table 2, row 3). However, deletion of nusG in ing ATP to examine the effect of NusABs and NusGBs on the the ⌬mtrB background reduced expression nearly 2-fold (Table accessibility of the ntDNA strand at the U107 and U144 pause 2, row 4). These results suggest that NusG only reduces expres- sites. Although every T residue was modified to a limited extent, sion when TRAP binds to the transcript. Thus, the 2- to 4-fold the T residues preceding U107 and U144 were particularly effect of nusG on TRAP-dependent regulation may underrep- reactive (Fig. 3). The intensity of footprint signals generally resent the effect of NusGBs-stimulated pausing on trp operon followed the kinetics of RNAPBs arriving to and escaping from expression. Experiments were also performed to determine the the pause sites observed in our transcription reactions (Fig. 2). effect of overexpressing nusA or nusG. Each gene was placed The footprint pattern Ϯ Nus factors at the U107 pause site were under control of a xylose-inducible promoter and introduced similar and extended from T95 to T107. The intensity of the basal into B. subtilis on a plasmid. Overexpression of nusG caused a footprint increased in the presence of NusABs but not NusGBs; 4-fold reduction of trpEЈ-ЈlacZ expression, whereas nusA over- however, NusGBs acted synergistically in combination with Nus- expression resulted in a more modest effect (Table 2, rows 5–10). ABs (Fig. 3). The signal between T120 and T129 observed in the Because our trpEЈ-ЈlacZ fusion studies suggested that NusGBs- absence of RNAP (data not shown) might be caused by localized stimulated pausing reduced expression of the trp operon, unwinding of this AT-rich region. The reactivity of this region KMnO4 footprinting was performed as a means to demonstrate was not affected by either Nus factor during transcription. directly that NusGBs promoted RNAP pausing at U144 in vivo. Because RNAPBs pauses at U107, the appearance of the The in vivo footprint of the WT nusG strain was distinct from the footprint at the U144 pause site with NusABs or both Nus factors in vitro footprint: every residue between 141 and 146 was was delayed, which reflects the increased time that it took modified, with T142 and T144 being the most reactive bases RNAPBs to reach U144 (Fig. 3). In each case, the footprint at the (compare Figs. 3 and 4). The permanganate reactivity gradually U144 pause site extended from T134 to T144 (Fig. 3). The basal disappeared with time, a hallmark of RNAP pause escape. The and NusABs-stimulated footprint was most intense in the up- footprint of the U144 pause site was virtually absent in the nusG stream region of the transcription bubble (T134 to T139). The mutant strain, whereas expression of nusG on a plasmid com- footprint pattern with NusGBs at the 15- and 30-s time points was plemented this defect (Fig. 4). These observations indicate that similar to the basal pattern; however, at the later 120- and 240-s NusGBs primarily drives RNAPBs pausing at U144 in vivo and is time points the reactivity of T137–T139 was reduced with a consistent with the in vitro transcription studies showing that the concomitant increase in T141 and T142 reactivity. The footprint NusGBs pause-stimulating activity was much stronger than the with both Nus factors had an intermediate pattern between those activity of NusABs. Moreover, these results establish that Nus- obtained with only NusABs or NusGBs. The time-dependent GBs-stimulated pausing at the trp pause site regulates expression change in reactivity of the ntDNA strand suggests that NusGBs of the trp operon.

16134 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0808842105 Yakhnin et al. Downloaded by guest on September 30, 2021 scribe past the terminator despite the presence of bound TRAP. Because bound TRAP does not influence pausing at U144 (9), RNAP will pause at U144 but will eventually resume transcrip- tion, resulting in formation of the trpE SD sequestering hairpin, which inhibits TrpE synthesis (Fig. 1). In situations where the AT promotes transcription readthrough, RNAP pausing at U144 presumably provides a second opportunity for TRAP to bind to the nascent transcript. If TRAP binds when RNAP is paused at

Fig. 4. Effect of NusGBs and NusABs on footprints of U144 paused transcrip- U144, continued transcription will result in formation of the trpE tion bubbles in vivo.WT(ϩ)ornusG knockout (Ϫ) strains contained or did not SD sequestering hairpin. Conversely, if TRAP fails to bind, contain (Ϫ) a xylose-inducible gene on a plasmid as shown. Cells were treated RNAP eventually resumes transcription, resulting in an RNA with KMnO4 at the times shown above each lane after rifampicin addition to conformation in which the trpE SD sequence is available for block reinitiation of RNAP. Primer extension reactions were performed on ribosome binding. plasmid DNA purified from KMnO4-treated cells. The positions of T135, T144 (pause site), and T156 are marked. Role of Nus Factors in Transcription Elongation. During nucleotide addition, movements of the trigger loop in the ␤Ј subunit assist Discussion NTP-stimulated translocation of RNAPEc (13, 15). Movement of In contrast to NusA, a pause-stimulating activity of NusG had the trigger loop is restricted at the his pause site, which contrib- not been observed in any organism. NusA and NusG syner- utes to stabilization of the PTC (13). Interaction of the pause Bs Bs hairpin with the ␤ flap domain of RNAP favors pausing by gistically stimulate pausing at U107 and U144 in vitro, resulting Ec inhibiting forward translocation of RNAP (12). NusA in- in 3- and 22-min half-lives, respectively (Fig. 2 and Table 1). Ec Ec creases the pause half-life by stimulating the basal interaction of However, pausing was not observed at U107 in vivo, whereas the the pause hairpin with the ␤ subunit (12, 13). In contrast, NusG in vivo U144 pause half-life is only Ϸ25 s (10). Transcription can Ec promotes forward translocation of RNAP during nucleotide involve movement of multiple RNAP molecules along the same Ec addition. Thus, NusG and NusA promote forward and gene in vivo, whereas only a single RNAP molecule is capable of Ec Ec backward translocation of RNAP , respectively, thereby mod- transcribing each template in single-round transcription reac- Ec ulating elongation in opposite directions (15). tions in vitro. Studies with RNAPEc have shown that a trailing NusGBs and NusGEc have different activities on transcription BIOCHEMISTRY RNAP molecule is capable of promoting forward translocation elongation in vitro. In particular, the strong RNAP pause- of a leading backtracked RNAP molecule, thereby relieving the Bs stimulating activity of NusGBs at the U144 pause site was not block to transcription elongation by a process referred to as observed with NusG irrespective of the source of RNAP. For RNAP cooperativity (14). Thus, RNAP cooperativity may re- Ec comparison, NusABs and NusAEc exhibited similar pause- duce the U144 pause half-life in vivo. The different temperatures stimulating activities at the U144 and E. coli his pause sites with used in the in vitro (23°C) and in vivo (37°C) experiments might both RNAPs. It was shown that NusG has little effect on also contribute to the difference in pause half-lives. Ec RNAPEc pausing at the his pause site and that this protein accelerates pause escape from a hairpin-independent ops pause Physiological Relevance of RNAP Pausing. The participation of site. In contrast, NusAEc enhances pausing at the his pause site NusABs and NusGBs in reducing expression of the B. subtilis trp and has little effect at the ops site (5). NusGEc and NusAEc also operon establishes the physiological relevance of their stimula- contribute to formation of RNAPEc antitermination complexes tory effect on RNAPBs pausing. Compared with NusABs, the (2), whereas NusGEc–Rho interaction facilitates transcript re- stronger influence of NusGBs on TRAP-dependent expression lease in Rho-dependent termination (6). Finally, NusGEc is (Table 2) is reflected in the in vitro and in vivo pausing known to accelerate the rate of transcription (2), and our results experiments (Figs. 2–4 and Table 1). The absence of NusG suggest that NusGBs may possess this general property as well. virtually eliminated pausing at U144 in vivo, whereas a plasmid Thus, bacterial Nus factors are quite flexible in their ability to carrying nusG complemented this defect (Fig. 4). Moreover, affect transcription, both positively and negatively, in response to deletion of nusG and nusG overexpression exhibited opposite specific signals in the nascent transcript and other protein effects on TRAP-dependent expression of the B. subtilis trp factors. The pause-stimulating effect of NusGBs at the trp pause operon (Table 2). The finding that deletion of nusG in a sites provides another example of this remarkable flexibility. TRAP-deficient background reduced expression nearly 2-fold NusAEc stimulates the basal interaction of the his pause may suggest that NusGBs has the general ability to accelerate hairpin with the ␤ subunit of RNAPEc (11). The finding that transcription, a known property of NusGEc (2). Thus, the NusABs and NusAEc were freely interchangeable in vitro suggests inhibitory effect of NusG on TRAP-dependent expression may that NusABs stimulates interaction of the U144 pause hairpin underrepresent the overall impact of NusGBs-stimulated pausing with the ␤ subunit of RNAPBs as well. The pause-stimulating on trp operon expression. activity of NusGBs at the trp pause sites is specific for RNAPBs. Because U107 is the nucleotide that just precedes the critical However, NusGEc can compete with NusGBs at the U144 pause overlap between the AT and T structures, RNAPBs pausing at site (Fig. 2D). Thus, although NusABs and NusAEc appear to rely U107 participates in the transcription attenuation mechanism, on common modules in RNAP for binding, some form of presumably by providing additional time for TRAP to bind to the species-specific interaction is required for NusGBs-stimulated nascent trp leader transcript. Bound TRAP prevents formation pausing. What could be responsible for this selectivity? of the AT and releases paused RNAPBs such that transcription E. coli RfaH is a paralog of NusGEc (Fig. S3). The antipausing can resume (9). As a consequence, formation of the T results in and antitermination activity of RfaH depends on sequence- transcription termination at G140 or U141 (Fig. 1). Although specific interaction with the ops element in the ntDNA strand, NusABs increases the termination efficiency (9), NusGBs does whereas the action of NusGEc is thought to be sequence- not (A.V.Y. and P.B., unpublished results). independent (16). The presence of only one NusG homolog in B. Because U144 is downstream from the trp leader termination subtilis may suggest that NusGBs fulfills certain functions of both sites, pausing at U144 participates in the trpE translational E. coli NusG and RfaH. The finding that NusGBs causes reduced control mechanism rather than in attenuation. Because tran- and increased permanganate reactivity of T137–T139 and T141– scription termination is never 100% efficient, RNAP can tran- T142, respectively (Fig. 3), indicates that NusGBs alters the

Yakhnin et al. PNAS ͉ October 21, 2008 ͉ vol. 105 ͉ no. 42 ͉ 16135 Downloaded by guest on September 30, 2021 accessibility of the ntDNA strand in the PTC, presumably by (Qiagen). His-tagged NusABs was purified as described in ref. 9. nusG was PCR-amplified from the B. subtilis chromosome and fused in-frame with the changing the conformation of RNAPBs and/or by contacting the N-terminal His tag in a pQE80L-derived vector (Qiagen). The resulting plasmid ntDNA strand itself. The possibility that NusGBs binds to the 6 ntDNA strand of the U144 PTC in a sequence-specific manner (pAY94) was used to overexpress nusG in E. coli by induction with IPTG. may simultaneously explain the absence of pause-stimulating His-tagged NusGBs was purified as for NusABs. activity of NusG at the his pause site and NusG at the U144 Bs Ec In Vitro Transcription. Single-round in vitro transcription reactions and data pause site (Table 1 and Table S1). The nucleotide sequence analysis were performed as described in ref. 10. Halted elongation complexes between the U107 pause hairpin and the U107 pause site containing a 29-nt transcript were formed in a reaction containing ATP and (AUGUUUAUUCU107) is similar to the corresponding sequence GTP (8 ␮M each), 2 ␮M UTP, and 1 ␮Ci of [␣-32P]UTP at 37°C (no CTP). of the U144 pause site (UUAUUUGUUAU144). Because this Elongation was resumed by the addition of all four NTPs together with 100 conservation includes the residues corresponding to T137–T139 ␮g/ml heparin at 23°C or 37°C. The final NTP concentrations were 150 ␮M CTP and T141–T142 of the U144 pause site (bold), it is possible that and UTP, and 10 ␮Mor150␮M ATP and GTP. Unless specified otherwise, the ␮ NusGBs makes sequence-specific contacts with the ntDNA concentration of NusABs, NusGBs, NusAEc, and NusGEc was 1 M. Aliquots of the strand at both pause sites. Although our studies have implicated transcription elongation reaction were removed at various times. Transcrip- tion of the last aliquot was continued for 10 min at 37°C with 0.5 mM each NTP. a role for the ntDNA in the mechanism of NusGBs action, it is likely that additional interactions between components of the Transcription reactions for KMnO4 footprinting were initiated on templates Ј 32 PTC and NusG play critical roles in the pausing mechanism. that were 5 end-labeled with P on the ntDNA strand. DTT was decreased to Bs 1 mM, and [␣-32P]UTP was omitted in these transcription reactions. The existence of hairpin-dependent, hairpin-independent, and promoter-proximal pause sites in E. coli (5, 17) supports the KMnO4 Footprinting. In vitro KMnO4 footprinting was performed as described notion that a variety of distinct structural changes in the elon- in ref. 10 except piperidine was removed from the DNA cleavage reactions by gating RNAP can result in the temporary failure to extend the evaporation. DNA pellets were washed with acetone and dried, followed by nascent transcript. Such different pausing events could be mod- two cycles of dH2O and evaporation. Dried pellets were dissolved in gel ulated by separate transcription factors in response to distinct loading buffer. physiological signals in many, if not all, organisms. Because the In vivo KMnO4 footprinting followed a published procedure with modifi- cellular abundance of NusGBs and RNAPBs is comparable (18), cations (10). Each strain contained pAY127 (trp leader). Some strains also the amount of NusG appears to be sufficient to bind essentially contained plasmid-encoded nusG, nusA, or vector. Cultures were grown at all elongating RNAP molecules. Thus, in conjunction with 37°C in minimal medium containing 0.2% acid casein hydrolysate, 0.5% NusA, NusG likely affects RNAP pausing at numerous sites in glycerol, chloramphenicol (10 ␮g/ml), and tetracycline (15 ␮g/ml) as appro- the B. subtilis genome. It is also reasonable to assume that these priate. Cultures were induced with 2% xylose at Klett 40, and cells were harvested 3–4 h later. Rifampicin (0.1 mg/ml) was added at the time of cell two general transcription elongation factors participate in addi- harvest. Ten milliliters of each culture was incubated with 1 ml of 110 mM tional regulatory events that have yet to be identified. KMnO4 for 1 min at 37°C. Reactions were quenched with 1 ml of stop solution [150 mM EDTA (pH 8.0), 150 mM sodium acetate (pH 4.5), 0.7 M ␤-mercapto- Materials and Methods ethanol, and 5% (vol/vol) glycerol] and placed on ice. Cells pellets were Plasmids and bacterial strains are described in SI Materials and Methods. suspended in 160 ␮l of STE buffer (10) and stored at Ϫ20°C until plasmid purification. Lysozyme (1 mg/ml) and RNase A (0.1 mg/ml) were added fol- ␤-Galactosidase Assays. B. subtilis strains were grown at 37°C in minimal lowed by incubation for 20 min at 37°C. SDS (0.5%) and proteinase K (0.05 medium containing 0.2% acid casein hydrolysate, 0.5% arabinose or glycerol, mg/ml) were added, and incubation was continued for1hat37°C. Cell lysates 5 ␮g/ml chloramphenicol Ϯ 1 mM tryptophan. Tetracycline (15 ␮g/ml) was were mixed with 250 ␮l of solution 3 from the StrataPrep kit (Stratagene), and added as appropriate. Expression of plasmid-encoded nusA and nusG was plasmids were purified according to the manufacturer’s protocol. Modified induced with 2% xylose during early exponential phase growth (Klett 40). bases were mapped by primer extension with Sequenase (USB Corp.) with an ␤ -Galactosidase activity was determined as described in ref. 10. oligonucleotide that annealed to bases 212–232 of the trp leader ntDNA strand. Additional details are SI Materials and Methods. DNA Templates and Proteins. DNA templates for in vitro transcription of the E. coli his leader or WT and mutant (U144A) B. subtilis trp leaders were obtained ACKNOWLEDGMENTS. We thank Cathy Squires (Stanford University, Stan- by PCR amplification using pAY66, pAY86, and pAY76 as templates, respec- ford, CA) and Mikhail Kashlev (National Cancer Institute, Frederick, MD) for ␴A ␤Ј tively. -containing RNAPBs with a His-tagged subunit was purified on providing NusAEc and NusGEc, respectively, and Robert Landick for advice. This Ni-nitrilotriacetic acid–agarose according to the manufacturer’s protocol work was supported by National Institutes of Health Grant GM52840.

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