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Rapa, a Bacterial Homolog of SWI2/SNF2, Stimulates RNA Polymerase Recycling in Transcription

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Rapa, a Bacterial Homolog of SWI2/SNF2, Stimulates RNA Polymerase Recycling in Transcription Downloaded from genesdev.cshlp.org on October 4, 2021 - Published by Cold Spring Harbor Laboratory Press RapA, a bacterial homolog of SWI2/SNF2, stimulates RNA polymerase recycling in transcription Maxim V. Sukhodolets, Julio E. Cabrera, Huijun Zhi, and Ding Jun Jin1 Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA We report that RapA, an Escherichia coli RNA polymerase (RNAP)-associated homolog of SWI2/SNF2, is capable of dramatic activation of RNA synthesis. The RapA-mediated transcriptional activation in vitro depends on supercoiled DNA and high salt concentrations, a condition that is likely to render the DNA superhelix tightly compacted. Moreover, RapA activates transcription by stimulating RNAP recycling. Mutational analyses indicate that the ATPase activity of RapA is essential for its function as a transcriptional activator, and a rapA null mutant exhibits a growth defect on nutrient plates containing high salt concentrations in vivo. Thus, RapA acts as a general transcription factor and an integral component of the transcription machinery. The mode of action of RapA in remodeling posttranscription or posttermination complexes is discussed. [Key Words: RapA; SWI2/SNF2 homolog; transcriptional activation; RNA polymerase recycling; remodeling posttranscription complexes] Received August 10, 2001; revised version accepted October 17, 2001. In Escherichia coli, core RNA polymerase (RNAP), Muchardt and Yaniv 1999). These proteins are capable of ␣ ␤␤Ј which consists of subunits 2 , is capable of transcrip- altering the configuration of naked DNA, an activity tion elongation and termination at simple terminators. that may be responsible for their chromatin/nucleosome On a sigma factor binding to core RNAP, the resulting remodeling function (Havas et al. 2000; Gavin et al. RNAP holoenzyme can initiate transcription at promot- 2001). ers on a DNA template (Burgess et al. 1969, 1987). There We found that RapA binds to both core RNAP and are also multiple RNAP-associated proteins, such as RNAP holoenzyme, with a higher affinity for the former, NusA, GreA/GreB and ␻, which on binding to RNAP at the interface of the ␣ and ␤Ј subunits (Sukhodolets and (core and/or holoenzyme) affect various steps in the tran- Jin 2000). Like other members of the SWI2/SNF2 protein scription cycle or RNAP assembly (Greenblatt and Li family, RapA is an ATPase. The ATPase activity of RapA 1981; Friedman and Gottesman 1983; Sparkowski and is stimulated on binding to RNAP, indicating that RapA Das 1991; Borukhov et al. 1993; Altman et al. 1994; Feng interacts with RNAP both physically and functionally. et al. 1994; Hsu et al. 1995; Mukherjee and Chatterji However, we found no apparent effect of rapA on cell 1997; Minakhin et al. 2001). growth in vivo and observed only a marginal effect of Previously, we identified an E. coli RNAP-associated RapA on transcription in vitro (Sukhodolets and Jin protein named RapA (Sukhodolets and Jin 1998). This 1998). Furthermore, our results (Sukhodolets and Jin 110-kD protein (also known as HepA) was also reported 2000) indicated that rapA is not likely to be involved in independently by another group (Muzzin et al. 1998). DNA repair, contrary to a report that a mutation in the RapA is a member of the SWI/SNF superfamily of heli- gene causes UV sensitivity (Muzzin et al. 1998). case-like proteins, which share six evolutionarily con- To search for the function of RapA in transcription, we served regions (Carlson et al. 1984; Andrews and Her- reasoned that because RapA is a bacterial homolog of skowitz 1989; Lewis et al. 1992; Bork and Koonin 1993; SWI2/SNF2, it is conceivable that it may retain the in- Kolsto et al. 1993; Eisen et al. 1995). Eukaryotic mem- trinsic ability to modulate DNA conformation, leading bers of this superfamily are implicated in chromatin/ to regulation of transcription. It is possible that RapA nucleosome remodeling and gene expression (for re- prefers a particular DNA conformation to act on in tran- views, see Peterson 1996; Pazin and Kadonaga 1997; scription. It is known that salt concentration has dra- matic effects on the conformation of supercoiled DNA (Bednar et al. 1994; Rybenkov et al. 1997a,b). In this re- 1Corresponding author. port we have determined the effect of RapA on transcrip- E-MAIL [email protected]; FAX (301) 594-3611. Article and publication are at http://www.genesdev.org/cgi/doi/10.1101/ tion of supercoiled DNA at different salt concentrations gad.936701. and found that it greatly stimulates transcription at rela- 3330 GENES & DEVELOPMENT 15:3330–3341 © 2001 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/01 $5.00; www.genesdev.org Downloaded from genesdev.cshlp.org on October 4, 2021 - Published by Cold Spring Harbor Laboratory Press RNAP recycling in transcription by RapA tively high salt concentrations. Our results demonstrate of the stimulation was several-fold lower than that ob- that RapA is a general transcriptional activator impor- served with RapA. Thus, RapA is a potent transcrip- tant for RNAP recycling in transcription. tional activator at relatively high concentrations of NaCl. Similar results were obtained when KCl was used (Fig. 1B). Results Because potassium glutamate, rather than NaCl or KCl, is the major intracellular salt in E. coli (Cayley et al. RapA activates transcription in vitro 1991), the effect of this salt on the ability of RapA to We decided to investigate the effect of RapA on tran- activate transcription was determined (Fig. 1C). RapA scription as a function of salt concentration using a su- activated transcription of tac significantly only when po- percoiled DNA containing the tac promoter (pDJ631) as tassium glutamate concentrations were Ն400 mM, with DNA template (Fig. 1). We found that the magnitude of maximal stimulatory effects at ∼500 mM potassium glu- the effect of RapA on transcription is dramatically influ- tamate. The consistent behavior of RapA in the presence enced by salt concentration (Fig. 1A). Specifically, RapA of different kinds of salt indicates that RapA generally had a strong stimulatory effect when NaCl was higher requires a relatively high salt concentration to stimulate than 100 mM; the activation reached its maximum at transcription. 200 mM NaCl. At relatively low (Յ100 mM) or very high The ability of RapA to activate transcription depends (300 mM) salt concentrations, RapA had only a minimal on a supercoiled DNA template. When the same plasmid effect on transcription. In the absence of RapA, transcrip- DNA was linearized, transcription was more sensitive to tion from the tac promoter was also sensitive to salt salt concentration overall than that on supercoiled DNA concentration. The transcription was enhanced when (at Ն250 mM NaCl RNAP nearly ceased synthesis of the the NaCl concentration was increased up to 250 mM and tac transcript), and RapA exhibited an inhibitory effect then declined at 300 mM NaCl. However, the magnitude at all salt concentrations tested (Fig. 2). Figure 1. RapA stimulates transcription on supercoiled DNA in the presence of high con- centration of salts. in vitro transcription reac- tions were carried out as described in Materi- als and Methods. Autoradiographs of the in vitro transcription gels showing the termi- nated tac transcript are presented. The NTP concentrations (A/U/G/C) were 1/0.2/0.2/ 0.02 mM, respectively. The concentrations of RNAP holoenzyme, RapA (purified from the 1:1 RNAP holoenzyme–RapA complex), and supercoiled plasmid DNA template (pDJ631) were approximately 12 nM, 0.4 µM (when present), and 12 nM, respectively. The effect of RapA on transcription from the tac pro- moter as a function of: NaCl concentration (A), KCl concentration (B), and potassium glu- tamate (KGlu) concentration (C). The quanti- tated levels of the tac transcript in reactions with (solid symbols) or without (open sym- bols) RapA are shown at right. GENES & DEVELOPMENT 3331 Downloaded from genesdev.cshlp.org on October 4, 2021 - Published by Cold Spring Harbor Laboratory Press Sukhodolets et al. Figure 2. RapA fails to stimulate transcription on linear DNA. The experimental conditions were similar to those described in Figure 1A, ex- cept that the purified 1.2-kb BfaI DNA fragment of pDJ631 (the BfaI sites are located ∼900 bp up- stream and ∼300 bp downstream from the tac pro- moter in the plasmid) at the concentration of ∼5 nM was used as a DNA template. The quantitated levels of the tac transcript in reactions with (solid symbols) or without (open symbols) RapA are shown at right. To rule out the possibility that RapA-mediated tran- motif Ia RapA mutant) difference in the affinity of the scriptional activation was promoter-specific, we deter- two mutant enzymes to RNAP compared to recombi- mined the effect of RapA on several other promoters (Fig. nant wild-type RapA using a glycerol gradient ultracen- 3). RapA was capable of stimulating transcription at all trifugation-binding assay (Sukhodolets and Jin 2000). the promoters tested when a relatively high concentra- However, the affinity of recombinant wild-type RapA to tion of salt was present in the reaction (under the con- RNAP was reduced ∼5- to 10-fold compared to that of ditions used, RapA increased RNA synthesis 4- to 20- wild-type (endogenous) RapA purified from the 1:1 fold compared to RNAP alone), although each promoter RNAP–RapA complex (data not shown). had its own characteristic profile. In the case of the strin- We then tested the effects of the two mutant RapA gent promoters rrnB P1 or pyrBI, there was no transcrip- proteins on transcription in the presence of high concen- tion at 300 mM NaCl either in the presence or absence of trations of NaCl. For the recombinant wild-type RapA RapA, reflecting the intrinsic instability of initiation protein, it showed stimulatory effect on transcription of complexes on stringent promoters (Gourse 1988; Zhou tac at a molar ratio (RapA/RNAP) 1; the stimulatory ef- and Jin 1998).
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