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Previous biochemical studies have suggested a role for bacterial DNA topoisomerase (TOPO) I in the suppression of R-loop formation during transcription. In this report, we present several pieces ofgenetic evidence to support a model in which R-loop formation is dynamically regulated during transcription by activities of multiple DNA TOPOs and RNase H. In addition, our results suggest that events leading to the serious growth problems in the absence of DNA TOPO I are linked to R-loop formation. We show that the overexpression ofRNase H, an enzyme that degrades the RNA moiety of an R loop, can partially compensate for the absence of DNA TOPO I. We also note that a defect in DNA gyrase can correct several phenotypes associated with a mutation in the rnhA gene, which encodes the major RNase H activity. In addition, we found that a combination of topA and rnhA mutations is lethal.DNA topoisomerase (TOPO) I, originally known as o, is the major DNA relaxing activity in Escherichia coli (1). The gene encoding E. coli DNA TOPO I topA was localized to the cys-trp region of the chromosome (2, 3). Deletions that remove both cysB and topA were identified in E. coli strains and it was first assumed that topA was not an essential gene (3). A more careful analysis showed that a deletion of the topA gene could only be inherited in strains that contained a second mutation that would compensate for the loss of topA (4, 5). These studies identified mutations in both gyrA and gyrB that could compensate for the loss of topA and demonstrated that these mutations result in reduced global DNA supercoiling. This lead to a picture in which a "global balance" of DNA supercoiling is essential and controlled by the competing activities of DNA gyrase and TOPO I.In vitro DNA TOPO I does not relax negatively supercoiled DNA to completion (1). Other in vitro studies show that the specificity for negatively supercoiled DNA derives from a requirement of a short single-stranded DNA region as part of the enzyme-DNA complex (6). Since negative DNA supercoiling favors the unpairing of DNA strands, DNA molecules with high negative superhelical density will be a better substrate for DNA TOPO I. Some in vivo studies also suggest that DNA TOPO I does not relax DNA efficiently: in one study a topA mutation had little effect on the rate of in vivo DNA relaxation after the inhibition of DNA gyrase by coumermycin (7); and in a second in vivo study, DNA TOPO I produced only slow and partial DNA relaxation (8). However, a defect in DNA TOPO I can cause an in vivo increase in the level of negative supercoiling (5). Thus, these results suggest that DNA TOPO I functions to prevent DNA supercoiling from reaching an unacceptably high level.The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 3526Hypernegatively supercoiled DNA can, under certain circumstances, be produced during transcription (9, 10). In...

We have previously shown that the genes encoding the two subunits of Escherichia coli DNA gyrase are regulated in a manner which is dependent on DNA conformation. When the DNA encoding the gyrA and gyrB genes is relaxed, both genes are expressed at a high level; in negatively supercolled DNA they are expressed at a low level. In this paper we describe fusions of both the gyrA and gyrB S' sequences to the E. coli galactokinase gene. In such fusions we found that galactokinase can be induced by treating the cells with coumermycin Al, an inhibitor of DNA gyrase. Our results suggest that the regulation occurs at the transcriptional level and that only a small region of DNA is necessary for coumermycin-induced gene expression.Covalently closed DNA isolated from eubacteria is known to be negatively supercoiled (4,9). If the supercoiling activity of DNA gyrase is blocked, cell growth is arrested (7,11). Furthermore, the degree of supercoiling must be maintained in a suitable range. In Escherichia coli, studies have implied that excessive DNA supercoiling also inhibits cell growth (5, 18). It has been well documented that supercoiling is important to the DNA functions of replication, transcription, and certain types of recombination (see references 6 and 9 for reviews).Given this background, we an-ticipated that there is somne sort of control over the expression of the genes encoding DNA gyrase, the enzyme responsible for DNA supercoiling. Our initial studies demonstrated that the gyrA and gyrB genes are directly regulated by the level of DNA supercoiling, in a manner appropriate to the function of DNA gyrase. When DNA is relaxed, synthesis of the subunits of DNA gyrase increases, thus enhancing the capacity of the cells to supercoil their DNA. Conversely, when DNA is supercoiled, expression of the genes encoding DNA gyrase decreases.From the standpoint of cellular regulation, the control of gyrase expression is readily understandable. However, the mechanism by which DNA relaxation activates transcription is not clear. Studies have demonstrated a mechanism for activation of transcription by DNA supercoiling. An early step in the initiation of transcription is the unwinding of the DNA by RNA polymerase to form the "open complex" (for a review see reference 13). This step is facilitated by negative supercoiling, which lowers the free energy needed to unwind DNA (8 DNA relaxation. Thus, such promoters are interesting objects of investigation.As a next step in our study of the control of gyrase expression and the nature of relaxation-activated gene expression, we constructed operon fusions between the gyrase genes and the structural gene for galactokinase. These fusions and their behavior are described in this paper.

Expression of the genes determining the subunits of Escherichia coli DNA gyrase (gyrA and gyrB) is known to be induced by relaxation of the template DNA. In this paper we report a deletion analysis of the gyrA and gyrB promoter regions. We find that a DNA sequence 20 base pairs long that includes the -10 consensus region, the transcription start point, and the first few transcribed bases is responsible for the property of induction by DNA relaxation. We propose a model for relaxation-stimulated transcription in which promoter clearance is the rate-limiting step.Gene expression in prokaryotes can be strongly influenced by changes in DNA supercoiling. In vivo experiments with inhibitors of DNA gyrase (EC 5.99.1.3) or with mutants defective in DNA topoisomerase I (EC 5.99.1.2) have shown that the transcription of various genes responds in different and characteristic ways when the extent of this supercoiling is altered (1-3). While production of some transcripts is much diminished when the DNA is relaxed, production of other transcripts is unaffected or even enhanced. The mechanism that couples DNA supercoiling to transcription is not well understood. Because negative DNA supercoiling favors the unwinding of the DNA double helix that is required in the formation of the RNA polymerase-DNA open complex, one could expect it to increase the rate of transcription, at least for those promoters for which open complex formation is rate limiting. Indeed, some experiments with purified RNA polymerase and supercoiled DNA have shown this effect; see, for example, the studies of the colicin El RNA I promoter by Wood and Lebowitz (4) and the lacPs promoter by Borowiec and Gralla (5). The cases in which transcription is enhanced by decreasing DNA supercoiling do not have such an easy explanation. This effect has been studied only in vivo (3, 6) or in multicomponent in vitro extracts (6); it is therefore unclear whether other factors besides RNA polymerase are involved and whether the required stretch of DNA extends beyond the promoter region.The present study addresses this latter question. Expression of the Escherichia coli gyrA and gyrB genes, which encode the subunits of DNA gyrase, is stimulated by DNA relaxation. The rates of synthesis of both subunits are increased approximately 10-fold in cells whose DNA gyrase activity is blocked or in crude extracts when the template DNA is presented in the relaxed form (6). The effect on in vivo expression of the gyrA and gyrB genes has been shown to be mediated at the transcriptional level (7). These genes are thus appropriate for the detailed study of relaxation-stimulated transcription (RST). We describe below the deletion analysis of the promoter structure of both genes. MATERIALS AND METHODSPlasmids, Strains, and Media. The deletion mutations described in this paper were generated from pGBK3 (gyrB) and pGAK1 (gyrA), which have been described elsewhere (7). The E. coli strains used in this study are derived from RM120 [str galK2 A(srl recA) srl:: TnJO] by introduction of the...

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