JOURNAL OF BACTERIOLOGY, Sept. 1988, p. 4266-4271 Vol. 170, No. 9 0021-9193/88/094266-06$02.00/0 Copyright © 1988, American Society for Microbiology Regulation of DNA Superhelicity by rpoB Mutations That Suppress Defective Rho-Mediated Termination in GAIL FERSTANDIG ARNOLDt AND IRWIN TESSMAN Department ofBiological Sciences, Purdue University, West Lafayette, Indiana 47907 Received 8 April 1988/Accepted 6 June 1988

The highly defective rho-IS mutant of Escherichia coli produces plasmid DNA that is 22% less negatively supercoiled than DNA from an isogenic wild-type strain (J. S. Fassler, G. F. Arnold, and I. Tessman, Mol. Gen. Genet. 204:424-429, 1986). We extended our measurements of plasmid superhelicity to additional rho mutants and to strains containing mutations that suppress rho transcription termination defects; the suppressor mutations were in the rpoB and the rho . The superhelicity of plasmid DNA was reduced by 11 and 10%, respectively, in the rho-702 and rho-201 mutants, both of which are less defective in Rho-mediated transcription termination than rho-15. Plasmid superhelicity was restored in all the suppressed rho mutants; in one rpoB mutant, plasmid DNA was even more negatively supercoiled than in rpoB+ cells, whether in a rho' or rho mutant background. Suppression of rho mutants enabled them to maintain plasmids that could not be maintained in the mutants in the absence of the suppressor mutations. The results indicate that in addition to DNA gyrase, topoisomerase I, and Rho, RNA polymerase is also a determinant of DNA superhelicity, and its effect is modified by the Rho protein. We propose that Rho may increase the degree of DNA unwinding by the transcription complex, possibly at transcription termination sites.

The ability of the Rho protein of Escherichia coli to affect RNA polymerase (40). Following the initial indications that DNA superhelicity in a substantial way provides the protein superhelical viral DNA can be more efficiently transcribed with a potentially powerful regulatory function and suggests than relaxed DNA both in vitro (25) and in vivo (42), much how some of the pleiotropy of the rho-15 might arise evidence that DNA superhelicity affects transcription initia- (12). The superhelicity of DNA, determined for the pBR327- tion has accumulated; most promoters are best transcribed related plasmid pJSF6, is reduced by 22 + 2% in rho-15 cells, from supercoiled templates, while some are best transcribed which is comparable to the reduction of 32 + 2% in from relaxed ones (for reviews, see references 10, 18, 53, gyrB203(Ts) gyrB22J(Cour) cells (12). The mechanism by and 54). A related observation is that mutations in the gene which Rho affects DNA superhelicity is not known. An for the d subunit of RNA polymerase (rpoB) can suppress indirect effect through the regulation of E. coli topoisomer- the defective initiation of transcription and replication pro- ase synthesis (12) now appears unlikely because cellular duced by a number of DNA gyrase mutations (11, 13, 35). levels of gyrase A, gyrase B, and topoisomerase I are not Furthermore, rpoB mutations and treatment of wild-type significantly altered in rho-15 cells (G. F. Arnold, Ph.D. strains with rifampin (which binds to the 1 subunit of RNA thesis, Purdue University, West Lafayette, Ind., 1987). We polymerase) can alter the degree of dependence of transcrip- focus on the possibility of a more direct effect of the Rho tion initiation on superhelicity (11, 13, 35). protein on superhelicity, which could conceivably occur RNA polymerase can unwind DNA during transcription through its activity in transcription termination. (17, 28, 44, 45). Here we report more lasting topological The aim of the current work was to see how DNA effects generated by the joint action of RNA polymerase and superhelicity is affected by mutations that suppress defective the Rho protein. The experiments described here show that Rho-mediated transcription termination. Some of the sup- all suppressors of the transcription termination defect of rho pressor mutations studied were in the rho gene itself, but the mutants, whether in the rpoB or rho gene, also suppress the most striking results were provided by suppressor mutations reduction in negative superhelicity. The suppressors also that were in the rpoB gene, which encodes the ,B subunit of correct for the plasmid maintenance deficiencies ofthese rho RNA polymerase. Transcription termination and the pausing mutants. We discuss how Rho and RNA polymerase acting that precedes it appear to be mediated by the ,B subunit of together could affect the superhelicity of DNA. RNA polymerase (14, 37, 56). Indirect genetic evidence (A preliminary presentation of this work has been made suggests that the Rho protein interacts with this subunit of [G. F. Arnold and I. Tessman, Abstr. Meet. Mol. Genet. RNA polymerase (9, 19, 21, 22, 57; S. Malik and A. and Phages, p. 216, 1987].) Goldfarb, Abstr. Meet. Phage and Bacterial Regulatory Mechanisms, p. 50, 1983). Furthermore, Rho has been MATERIALS AND METHODS shown to bind to the NusA protein (46), which in turn is known to bind to the 1B subunit of RNA polymerase (20, 46). Bacterial strains. The strains used in these studies are Like transcription termination and pausing, transcription listed in Table 1. initiation also appears to be mediated by the d subunit of Media. Bacterial cultures were routinely grown either in TB (13 g of tryptone [Difco Laboratories, Detroit, Mich.] and 7 g of NaCl per liter) supplemented with 50 ,ug of t Present address: Center for Advanced Biotechnology and Med- ampicillin per ml for transformed cultures or on LB plates icine, and Rutgers University, P.O. Box 759, Piscataway, NJ 08854. (34) with 5 g of NaCl per liter instead of 10 g/liter. The 4266 VOL. 170, 1988 DNA SUPERHELICITY ALTERED BY rpoB MUTATIONS 4267

TABLE 1. Bacterial strains Strain Relevant genotype Source (reference) IT1011 galP3 rho' rpoB+ SA1030 from A. Das (8) IT1022 As IT1011, but ilvY::TnIO This laboratory (51) IT1631 As IT1022, but rho-15 This laboratory (12) GFA261 As IT1022, but Tets Tets from IT1022a N4938 rpoBI01 S. Garges (9) GFA268 As GFA261, but rpoB1OJ Rif from GFA261 x P1(N4938) GFA270 As IT1631, but rpoBI01 Rif from 1T1631 x P1(N4938) GFA156 rho' rpoB+ A(tonB-IacP) X8605 from L. Guarente (22) GFA158 As GFA156, but rho-201 L. Guarente (22) GFA160 As GFA156, but rho-201 rpoB203 L. Guarente (22) GFA280 As GFA156, but rpoB203 Tetr from GFA160 x Pl(IT1022) H12007 rho' M. Imai (52) KT5375 As H12007, but rho-702 M. Imai (52) KT5376 As H12007, but rho'-77 M. Imai (52) KT5377 As H12007, but rho'-78 M. Imai (52) KT5378 As H12007, but rho'-81 M. Imai (52) KT5380 As H12007, but rho'-82 M. Imai (52) a Procedure of Maloy and Nunn (32).

scoring of defective Rho-dependent transcription termina- otherwise like its wild-type parent in being Ts', motile, and tion involved the use of LB-0 plates (LB plates without proficient at transcription termination (9). NaCl), MacConkey- plates (Difco), X-Gal plates The rpoB203 mutation was selected from a population of (34), LB plates supplemented with 10 ,ug of rifampin, and rho-201 cells by the ability of the double mutant to survive in gradient plates with 7.5 mg of novobiocin per ml by the the presence of rifampin and to show improved transcription method of Szybalski and Bryson (50). termination at the trp terminators (22). The rpoB203 rho, rpoB, and gyrB mutants. rho-15 mutants were identi- mutant is also temperature sensitive (like the rho-201 fied by six criteria: formation of red colonies on MacConkey- rpoB203 double mutant). The rpoB203 mutation is notable in galactose plates as a result of their transcription termination that it also corrects the transcription termination defects defect at the polar galP3 mutation (8); temperature sensitiv- conferred by other rho mutations, including a rho amber ity (8); rifampin supersensitivity (24); novobiocin supersen- mutation that is lethal in an rpoB+ background in the sitivity (unpublished data); nonmotility (S. Garges and S. absence of an amber suppressor (22). Although the RpoB203 Adhya, Abstr. Annu. Meet. Am. Soc. Microbiol. 1982, p. protein has been found to require Rho protein for termina- 115); and reduced growth rate. At the permissive tempera- tion at the highly Rho-dependent trp t' in vitro ture of 32°C, the rho-15 mutant shows roughly a 33-fold (55), that study was done in the absence of the apparently increase of transcription readthrough into the (8). important NusA protein, which binds to RNA polymerase The other rho mutants studied are all less defective in (20, 46) and Rho (46). Together, these features of the transcription termination than the rho-iS mutant. When RpoB203 protein suggest that it may be capable of transcrip- mutant alleles were transduced into the galP3 background, tion termination at most, though possibly not all, Rho- no red colonies were found, while other appropriate Rho- dependent terminators, even in the absence ofwild-type Rho phenotypes were seen to cotransduce with Ilv+ or Tetr protein. markers with the expected frequency of 50%. The less Temperature-resistant revertants of the rho-702 allele ca- defective rho mutants are all sensitive to rifampin and pable of terminating transcription better than rho' cells at novobiocin, although, as described below, they differ in their polar mutations in the trp and lac were named super motility and temperature sensitivity characteristics. rho (or rhos) strains (52). Like their wild-type parent, they The rho-201 mutation allowed cells to grow well at 42°C. can all grow at 42°C, but unlike their wild-type parent, they Nonmotility could not be used as an indicator for this strain are all nonmotile. The rho'-78 strain is slightly more resistant because the rho' parent was also nonmotile. At an unspec- to rifampin than the rho' parent. ified temperature, the rho-201 mutation confers a 10- to Transformation, plasmid preparation, and superhelicity 47-fold increase in transcription readthrough at the end of determinations. The methods were as described elsewhere the in E. coli (22, 23). We confirmed the presence (12). Superhelicity is presented in terms of the difference of the transcription termination defect for our rho-201 strain, (AL) between the average linking number of a plasmid GFA158, by observing that transcription readthrough from isolated from a mutant strain and that of a control plasmid the trp operon into the yielded dark blue colonies isolated from the wild-type strain. on X-Gal plates in contrast to the pale blue colonies pro- duced by the rho' strain, GFA156. RESULTS The rho-702 mutant (previously called nitA702), unlike its parent, is both temperature sensitive (27) and nonmotile. The techniques we used to analyze DNA superhelicity The rho-702 mutation causes a roughly 40% reduction in have been described previously (12). In brief, plasmid transcription termination at several Rho-dependent tran- pJSF6, which had been derived by spontaneous mutation scription terminators at the permissive temperature of 300C from a variant of pBR327, was used because it can be (47). The transcription termination defect is significantly maintained in Rho-defective cells. The superhelicity was more severe at higher temperatures. determined from the position of the peak of the Gaussian The rpoB101 mutant is rifampin resistant (50 ,ug/ml) but is distribution of topoisomers that is formed after agarose gel 4268 ARNOLD AND TESSMANJ.BCEILJ. BACTERIOL.

TABLE 2. Changes in the linking number of pJSF6 DNA N produced by rho and rpo mutations

I /, L\", rho' rpoB' 5) standard and Genotype ALa ALrb 0C Z, strain 1T1022 (5)C IT1631 (4) rho-1S +4.0 ± 1.1 +22 ± 6 GFA268 (4) rpoB101 +0.3 ± 0.1 +2 ± 1 GFA27O (5) rho-iS rpoB101 +0.9 ± 0.1 +5 ± 1 GFA156 (4) GFA158 (5) rho-201 +1.8 ± 0.3 +10 ± 2 GFA280 (5) rpoB2O3 1-1.2 ± 0.2 -6 ± 1 GFA16O (4) rho-201 rpoB2O3 -1.7 ± 0.3 -9 ± 2 H12007 (5) KT5375 (5) rho-702 +2.0 ± 0.1 +11 ± 1 KT5376 (4) rho'-77 +0.1 ± 0.0 +0.5 ± 0.0 KT5377 (5) rhoS-78 -0.1 ± 0.0 -0.5 ± 0.0 KT5378 (5) rhoS-81 +0.4 ± 0.0 +2 ± 0.0 KT5380 (4) rho'-82 +0.1 ± 0.0 +0.5 ± 0.0 " AL was calculated from peak ladder positions relative to those obtained from the isogenic wild-type standard strains, 1T1022, GFA156, and HI2007, as indicated. A positive value indicates that the DNA is less negatively super- coiled than the standard, that is, more relaxed; a negative value indicates that FIG. 1. An example of distributions of pJSF6 extracted from it is more negatively supercoiled. Errors are standard errors of the mean. various E. coli strains: rho' rpoB' (IT1022), rho-iS (IT1631), b Percent change in superhelicity of pJSF6 relative to the assumed value of rpoBlOl (GFA268), and rho-IS rpoBlO1 (GFA27O). Chloroquine, 5.0 18.5 supercoils in pJSF6 isolated from the wild-type parent strain 1T1022 at p.g/ml. 30'C (12). Errors are standard errors of the mean. The number of independent plasmid isolates analyzed in each case is indicated in parentheses. electrophoresis with chloroquine. Figure 1 shows examples of ladders produced by independent extracts of plasmid DNA from wild-type and three mutant strains of E. coli. For the rpoB2O3 mutation alone could produce a 6 ± 1% increase a plasmid grown in a particular mutant and the plasmid in superhelicity over the wild-type parent strain (rho' grown in the reference wild-type cell, the difference between rpoB'); it indicates that RNA polymerase is capable not their peak ladder positions gives the difference (AL) between only of compensating for Rho defects but also of causing their linking numbers. The relative change in the linking DNA superhelicity to be altered even in a rho' cell. number (AL,) was calculated by assuming that pJSF6 grown Effects of four super rho mutations in the rho-702 mutant. in wild-type cells at 300C is underwound by an average of The rho-702 mutation reduced the superhelicity of pJSF6 18.5 turns. DNA by 11 ± 1% (Table 2). The four rhos mutations, which Effects of the rpoBlOl mutation on the rho-iS mutant. We are characterized by the ability to terminate transcription confirmed the previous observation (12) that the highly better than rho' cells at several Rho-dependent transcrip- deleterious rho-iS mutation produces a 22 ±4 2% reduction in tion terminators (47), restored the superhelicity to within 0.5 the negative superhelicity of the plasmid pJSF6 at 300C to 2% of the value obtained for the rho' strain. (Table 2). The mutation rpoB 101 in the P3 subunit of RNA Plasmid maintenance in the suppressed rho strains. Many polymerase suppresses the rho-iS mutation to the extent that plasmids cannot be maintained in some rho mutants, such as the rho-iS rpoB 101 double mutant shows essentially wild- rho-iS and rho-201 (2, 12, 30; S. E. Brown, Ph.D. thesis, type levels of transcription termination at 300C (9), which we Harvard University, Cambridge, Mass., 1982; J. S. Fassler, confirmed by the white colonies it produced on MacConkey- Ph.D. thesis, Purdue University, West Lafayette, Ind., 1983; galactose plates at 300C. In contrast to the 22% reduction in unpublished data). Included among these plasmids are plasmid superhelicity found in the rho-IS strain, the double pBR322 and a plasmid originally described to us as pBR327 mutant showed only a 5 ±+- 1% reduction. Thus, the rpolli0i but different from other sources of pBR327 in that it could mutation restored the superhelicity to nearly the wild-type not be maintained in rho-iS cells (12). We refer to this level. The rpoBi0i mutation alone had little effect on plas- mutant plasmid as pBR327m; it is from this plasmid that mid superhelicity, producing an insignificant 2 ± 1% reduc- pJSF6, which can be maintained in rho-iS cells, was isolated tion. in one step (12). Suppression of the transcription termination Effects of the rpoB2O3 mutation on the rho-201 mutant. The and superhelicity deficiencies of the rho-iS and rho-20J rho -201 mutation reduced the negative superhelicity of mutants by the rpoB101 and rpoB2O3 mutations, respec- pJSF6 DNA by 10 ± 2% (Table 2). The transcriptional tively, also suppressed the plasmid maintenance deficiency readthrough of the trp operon terminators caused by the (Table 3). Surprisingly, the rho-702 strain was able to main- rho-20i mutation is almost completely suppressed by the tain pBR322 even though it was quantitatively like rho-201 in mutation rpoB2O3 (22, 23); in our hands the double mutant its reduction of pJSF6 superhelicity (Table 2). produced a white colony on X-Gal plates in contrast to the dark blue color of the rho-20i mutant, confirming suppres- DISCUSSION sion of the transcription termination defect. We observed that the superhelicity of the plasmid DNA from the rho -201 We previously reported that plasmid DNA purified from mutant was not only restored by the rpoB2O3 mutation but the highly defective rho-iS mutant displays negative super- was even 9 ± 2% greater than that produced by the wild-type helicity that is 22% less than that of plasmid DNA purified (rho' rpoB') parent strain. It is particularly significant that from an isogenic rho' strain (12); the mutant DNA is more VOL. 170, 1988 DNA SUPERHELICITY ALTERED BY rpoB MUTATIONS 4269

TABLE 3. Maintenance of plasmids pBR322 and pBR327m in DNA, which could then provoke subsequent action by E. coli strains topoisomerases (12). The involvement of Rho in the tran- Result for plasmida: scription process provides an opportunity for such a direct Strain Genotype effect. The unwinding of the DNA duplex during transcrip- pBR322 pBR327m tion should generate underwound (negatively supercoiled) IT1022 Wild type + + DNA upstream of the complex and overwound (positively IT1631 rho-15 - - supercoiled) DNA downstream (31). Positive superhelical GFA270 rho-15 rpoB101 + + turns, which hinder the transcription process, are thought to be normally removed, at least in part, by reduction of the GFA156 Wild type + + linking number through the action of DNA gyrase (10, 18, GFA158 rho-201 - + 54), and that would affect the net superhelical density of the GFA160 rho-201 rpoB203 + + entire DNA molecule. Thus, if at some stage of transcription to H12007 Wild type + + the Rho protein acted enhance topoisomerase activity by KT5375 rho-702 + + DNA gyrase, it could explain why mutants deficient in Rho KT5376 rhos-77 + + activity have less negatively supercoiled DNA than their rho+ counterparts. a Cells were transformed at a frequency on the order of 10-6. +, Approx- One possibility is that Rho could cause the transcription imately 50 ,ug of plasmid DNA could be isolated from 5 x 10"' transformed to and more time for intro- no DNA could isolated. complex pause thereby provide (Apr) cells; -, plasmid be duction of negative superhelical turns by DNA gyrase before the transcription complex dissociates. (Two reports claim relaxed. Here we show that two more rho strains, rho-702 that Rho has no effect on pausing at the lambda tRl tran- and rho-201, produced significant reductions in negative scription terminator [29, 36], but those studies were per- supercoiling of 11 and 10%, respectively, indicating that the formed in vitro without inclusion of the NusA protein, relaxation effect is not peculiar to the rho-15 strain. Those whereas the terminator is preceded by a boxA binding site observations led us to consider whether some Rho-depen- for NusA [15].) A second possibility is that Rho could dent aspect oftranscription, such as the termination process, increase the degree to which the DNA duplex is unwound by might somehow affect the degree of DNA supercoiling. the transcription complex; corrective action taken down- Our approach was to examine the effect of suppressor stream would then increase the overall negative superhelic- mutations that restore Rho-mediated transcription termina- ity of the DNA. Because Rho is a termination factor, it could tion to each of the four rho strains; in particular we measured well be that itjoins RNA polymerase at termination sites (or the superhelicity of plasmids isolated from the suppressed at pause sites preparatory to termination) to produce the rho strains. The suppressor mutations map either in the rpoB enhanced unwinding effect. And although Rho is a necessary gene, which encodes the I subunit ofRNA polymerase, or in factor for termination only at sites termed Rho dependent, it the rho gene. The key observation was that for both classes remains an open question whether Rho might affect the of suppressor mutations, increased transcription termination function of RNA polymerase and thus also affect DNA was associated with increased negative superhelicity of the superhelicity by acting at so-called Rho-independent sites as plasmid DNA. well. We examined four super rho strains derived from the It has been reported that in vitro in the presence of ATP, rho-702 mutant; they were characterized by their ability to Rho can cause dissociation of short RNA-DNA heterodu- terminate transcription even better than the rho+ parent at plexes designed to mimic Rho-dependent transcription ter- several Rho-dependent transcription terminators (47). All minators (4). Those studies were done in the absence of rho' suppressors produced plasmnids that were negatively RNA polymerase and required much larger amounts of ATP supercoiled about as much as, but no more than, plasmids than are needed for transcription termination in vitro. Nev- from the isogenic rho' strain. When the rpoB101 mutation ertheless, such helicase-like activity might be another man- was combined with the rho-15 allele, it too restored the ifestation of the ability of Rho to unwind nucleic acid normal level of plasmid superhelicity. Notably, the rpoB203 duplexes and could be related to the ability of Rho to bind to mutation, which is known to suppress the transcription both single- and double-stranded DNA (3, 19, 39, 43). termination defect of numerous rho afleles (22), produced The sensitivity of rho mutants to the antibiotic rifampin, plasmids that had even more negative supercoils than plas- which binds to the ,B subunit of RNA polymerase, is consis- mids from wild-type cells, regardless of whether the rpoB203 tent with the idea that Rho and the polymerase act jointly to mutation was combined with the rho' or the rho-201 allele. influence the supelhelicity of the DNA. Rifampin treatment Thus, an rpoB mutation alone can alter DNA superhelicity. of wild-type cells promotes transcription readthrough, both From the data presented here we propose that Rho activ- at Rho-independent (26, 38) and at Rho-dependent (7) termi- ity is coupled to topoisomerase activity through the tran- nation sites. Many rho mutants are supersensitive to rifam- scription process. It has already been argued that the un- pin (24), including all those studied here (unpublished data), winding of the DNA duplex by RNA polymerase during but suppressed rho mutants (rho rpoB and rho rhos) show transcription is coupled to DNA gyrase activity in such a normal or greater than normal resistance to rifampin (9, 22; way that the transcription process itself determines to some unpublished data), Inasmuch as rifampin also produces extent the degree of supercoiling (1, 5, 31, 41; Arnold, Ph.D. abortive transcription initiation (48), we should remain open thesis). The ability of rpoB mutations to suppress at the same to the possibility that Rho-mediated effects on DNA super- time both the transcription termination deficiencies and the helicity may occur through the transcription initiation pro- superhelicity deficiencies of rho mutants suggests that Rho cess, perhaps coupled to upstream termination processes. actsjointly with RNA polymerase at some stage of transcrip- We also considered whether Rho might have an indirect tion to influence the degree of supercoiling. effect on DNA superhelicity (12), particularly because we We have suggested that Rho could have a direct effect on know that the rho-15 mutation greatly alters the expression DNA superhelicity by locally altering the helical twist of the of many genes (Arnold, Ph.D. thesis; S. Garges, personal 4270 ARNOLD AND TESSMAN J. BACTERIOL. communication), some of which could be responsible for 4. Brennan, C. A., A. J. Dombroski, and T, Platt. 1987. Transcrip- topoisomerase production or activity. In contrast to a pre- tion termination factor Rho is an RNA/DNA helicase. Cell 48: liminary observation (cited in reference 12), we now observe 945-952. A and B and DNA 5. BrilI, S. J., S. Dinardo, K. Voelkel-Meiman, and R. Sternglanz. that the levels of the DNA gyrase proteins 1987. Need for DNA topoisomerase activity as a swivel for topoisomerase I are normal in rho-iS cells (Arnold, Ph.D. DNA replication for transcription of ribosomal RNA. Nature thesis), but there may be topoisomerase effectors that have (London) 326:414-416. altered expression in rho mutants. It is also conceivable that 6. Bujard, H., D. Stueber, R. Gentz, U. Deuschle, and U. Peschke. Rho itself has topoisomerase activity, but no evidence of 1984. Insertion of transcriptional elements outside the replica- that has been reported. tion region can interfere with replication, maintenance, and We have observed that rpoB mutants (in either rho' or rho stability of ColEl-derived plasmids, p. 45-52. In JD. R. Helinski, mutant backgrounds) and rho mutants (including rhos) are S. N. Cohen, D. B. Clewell, D. A. Jacksoin, and A. Hollaender hypersensitive to the DNA gyrase inhibitor novobiocin; (ed.), Plasmids in bacteria. Plenum Publishing Corp., New mutation York. furthermore, when the gyrB203(Ts) gyrB22i(Cou) 7. Cromie, K. D., and R. S. Hayward. 1984. Evidence for rifampi- (33) is introduced into the rho' strain IT1022, the strain cin-promoted readthrough of a fully Rho-dependent transcrip- becomes hypersensitive to rifampin (unpublished data). tional terminator. Mol. Gen. Genet. 193:532-534. Thus, although we must consider the possibility that the 8. Das, A., D. Court, and S. Adhya. 1976. Isolation and character- mutations increase membrane permeability to novobiocin ization of conditional lethal mutants of Escherichia coli defec- and rifampin, the observations are consistent with the view tive in transcription termination factor rho. Proc. Natl. Acad. that the Rho, RNA polymerase, and gyrase proteins are Sci. USA 73:1959-1963. mutually involved in! determining the degree of DNA super- 9. Das, A., C. Merril, and S. Adhya. 1978. Interaction of RNA polymerase and rho in transcription termination: coupled coiling. ATPase. Proc. Natl. Acad. Sci. USA 75:48284832. The ability to maintain plasmids pBR322 and pBR327m 10. Drlica, K. 1984. Biology of bacterial deoxyribonucleic acid was restored to rho mutants by the rpoB and rhos suppressor topoisomerases. Microbiol. Rev. 48:273-289. mutations. But since the suppressors increased both nega- 11. Ephrati-Elizur, E., and B. Chronis-Anner. 1984. Expression of tive superhelicity and transcription termination, we cannot silent genes: possible interaction between DNA gyrase and determine whether just one or both of these are necessary RNA polymerase, p. 435440; In U.. Hubbscher and S. Spadari for plasmid maintenance. Increased superhelicity might be (ed.), Proteins involved in DNA replication. Plenum Publishing needed for the binding of critical replication initiation pro- Corp., New York. teins (16); mote efficient transcription termination might be 12. Fassler, J. S., G. F. Arnold, and I. Tessiman. 1986. Reduced needed for critical transcription or replication events to superhelicity of plasmid DNA produced by the rho-15 mutation in coli. Mol. Gen. occur at the of of the The Escherichia Genet. 204:424429. origin replication plasmids (6, 49). 13. Filut6wicz, M., and P. Jonczyk. 1983. The gyrB gene product mutants rho-201 and rho-702 both reduced superhelicity by functions in both initiation and chain polymerization of Esche- about 110% (Table 2), yet rho-201 could not maintain pBR322, richia coli chromosome replication: suppression of the initiation while rho-702 could. Split-phenotype mutants having defec- deficiency in gyrB-ts mutants by a class of rpoB mutations. Mol. tive transcription termination but norinal superhelicity, if Geri. Genet. 191:282-287. they can exist, could provide insight into how Rho-mediated 14. Fisher, R. F., and C. Yanofsky. 1983. Mutations in the 3 subunit transcription termination itself might affect plasmid mainte- of RNA polymerase alter both transcription pausing and tran- nance as well as other aspects of cellular physiology. scription termhination in the trp operon leader region in vitro. J. While superhelicity regulates the transcription process, Biol. Chem. 258:8146-8150. we see that the reverse is also true and that both RNA 15. Friedman, D. I., and E. R. Olson. 1983. Evidence that a nucleotide sequence, "boxA," is involved in the action of the polymerase and Rho can influence DNA superhelicity. Al- NusA protein. Cell 34:143-149. though our meas.urements do not tell us whether the super- 16. FtanneIl, B. E., T. A. Baker, luid A. Kornberg. 1986. Complete helical tension is altered in vivo, the increased linking enzymatic replication of plasmids containing the origin of the number produced in vivo by the rho mutations may well Escherichia coli chromosome. J. Biol. Chem. 261:5616-5624. result in altered superhelical tension that could generate a 17. Gamper, H. B., and J. E. Hearst. 1982. A topological model for myriad of secondary effects, influencing such critical pro- transcription based on unwinding angle analysis of E. coli RNA cesses as , replication, recombination, re- polymerase binary, initiation and ternary complexes. Cell 29: pair, and DNA packaging. 81-90. 18. Gellert, M. 1981. DNA topoisomerases. 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