Marc Drolet

Growth inhibition mediated by excess negative supercoiling: the interplay between transcription elongation, R-loop formation and DNA topology

It has been known for a long time that supercoiling can affect gene expression at the level of promoter activity. Moreover, the results of a genome‐wide analysis have recently led to the proposal that supercoiling could play a role in the regulation of gene expression at this level by acting as a second messenger, relaying environmental signals to regulatory networks. Although evidence is lacking for a regulatory role of supercoiling following transcription initiation, recent results from both yeast and bacteria suggest that the effect of supercoiling on gene expression can be considerably more dramatic after this initiation step. Transcription‐induced supercoiling and its associated R‐loops seem to be involved in this effect. In this context, one major function of topoisomerases would be to prevent the generation of excess negative supercoiling by transcription elongation, to inhibit R‐loop formation and allow gene expression. This function would be especially evident when substantial and rapid gene expression is required for stress resistance, and it may explain, at least in part, why topoisomerase I synthesis is directed from stress‐induced promoters in Escherichia coli. Growth inhibition mediated by excess negative supercoiling might be related to this interplay between transcription elongation and supercoiling.

Escherichia coli DNA topoisomerase I inhibits R-loop formation by relaxing transcription-induced negative supercoiling.

It has recently been shown that RNase H overproduction can partially compensate for the growth defect due to the absence of DNA topoisomerase I in Escherichia coli(Drolet, M., Phoenix, P., Menzel, R., Massé, E., Liu, L. F., and Crouch, R. J. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 3526–3530). This result has suggested a model in which inhibitory R-loops occur during transcription in topAmutants. Results presented in this report further support this notion and demonstrate that transcription-induced supercoiling is involved in R-loop formation. First, we show that stable R-loop formation duringin vitro transcription with E. coli RNA polymerase only occurs in the presence of DNA gyrase. Second, extensive R-loop formation in vivo, revealed by the production of RNase H-sensitive hypernegatively supercoiled plasmid DNAs, is observed under conditions where topA mutants fail to grow. Furthermore, we have demonstrated that the coupling of transcription and translation in bacteria is an efficient way of preventing R-loop formation.

Relaxation of transcription-induced negative supercoiling is an essential function of Escherichia coli DNA topoisomerase I.

It has been suggested that the essential function of DNA topoisomerase I in Escherichia coli is to prevent chromosomal DNA from reaching an unacceptably high level of global negative supercoiling. However, other in vivo studies have shown that DNA topoisomerase I is very effective in removing local negative supercoiling generated during transcription elongation. To determine whether topoisomerase I is essential for controlling global or local DNA supercoiling, we have prepared a set of topAnull mutant strains in combination with different plasmid DNAs. Although we found a correlation between the severity of the growth defect with both transcription-induced and global supercoiling, near to complete growth inhibition correlated only with transcription-induced supercoiling. This result strongly suggests that the major function of DNA topoisomerase I is to relax local negative supercoiling generated during transcription elongation.

The problem of hypernegative supercoiling and R-loop formation in transcription.

DNA supercoiling and topoisomerases have long been known to affect transcription initiation. In many studies, topA mutants were used to perturb chromosomal supercoiling. Although such studies clearly revealed that supercoiling could significantly affect gene expression, they did not tell much about the essential function(s) of DNA topoisomerase I, encoded by topA. Indeed, the topA mutants used in these studies were growing relatively well, although this gene is normally essential for growth. These mutants were either carrying a topA allele with enough residual activity to permit growth, or if deleted for the topA gene, they were carrying a compensatory mutation allowing them to grow. We have recently used a set of isogenic strains carrying a conditional gyrB mutation that allowed us to study the real effects of losing topoisomerase I activity on cell physiology. The results of our work show that an essential function of topoisomerase I is related to transcription, more precisely to inhibit R-loop formation. This is in agreement with a series of biochemical studies that revealed a role for topoisomerase I in inhibiting R-loop formation during transcription in the presence of DNA gyrase. In addition, our studies may have revealed an important role for DNA supercoiling in modulating gene expression, not only at the level of transcription initiation but also during elongation. In this paper, we will first discuss global and local supercoiling, then we will address the topic of R-loop formation and finally, we will review the subject of hypersupercoiling and R-loop formation in gene expression. Whenever possible, we will try to make correlations with growth phenotypes, since such correlations reveal the essential function of DNA topoisomerase I.

ROLES OF DNA TOPOISOMERASES IN THE REGULATION OF R-LOOP FORMATION IN VITRO

We have recently found that stable R-loop formation occurs in vivo and in vitro when a portion of the Escherichia coli rrnB operon is transcribed preferentially in its physiological orientation. Our results also suggested that the formation of such structures was more frequent in topA mutants and was sensitive to the template DNA supercoiling level. In the present report we investigated in greater detail the involvement of DNA topoisomerases in this process. By using an in vitro transcription system with phage RNA polymerases, we found that hypernegative supercoiling of plasmid DNAs in the presence of DNA gyrase is totally abolished by RNase H, suggesting that extensive R-looping occurs during transcription in the presence of DNA gyrase. When RNase A is present, significant hypernegative supercoiling occurs only when the 567-base pair rrnB HindIII fragment is transcribed in its physiological orientation. This result suggests that more stable R-loops are being produced in this orientation. Our results also suggest that DNA gyrase can participate in the process of R-loop elongation. The strong transcription-induced relaxing activity of E. coli DNA topoisomerase I is shown to efficiently counteract the effect of DNA gyrase and thus inhibit extensive R-looping. In addition, we found that an R-looped plasmid DNA is a better substrate for relaxation by E. coli DNA topoisomerase I as compared with a non-R-looped substrate.

Effects of RNA polymerase modifications on transcription‐induced negative supercoiling and associated R‐loop formation

Transcription in the absence of topoisomerase I, but in the presence of DNA gyrase, can result in the formation of hypernegatively supercoiled DNA and associated R‐loops. In this paper, we have used several strategies to study the effects of elongation/termination properties of RNA polymerase on such transcription‐induced supercoiling. Effects on R‐loop formation were exacerbated when cells were exposed to translation inhibitors, a condition that stimulated the accumulation of R‐loop‐dependent hypernegative supercoiling. Translation inhibitors were not acting by decreasing (p)ppGpp levels as the absence of (p)ppGpp in spoT relA mutant strains had little effect on hypernegative supercoiling. However, an rpoB mutation leading to the accumulation of truncated RNAs considerably reduced R‐loop‐dependent hypernegative supercoiling. Transcription of an rrnB fragment preceded by a mutated and inactive boxA sequence to abolish the rrnB antitermination system also considerably reduced R‐loop‐dependent supercoiling. Taken together, our results indicate that RNA polymerase elongation/termination properties can have a major impact on R‐loop‐dependent supercoiling. We discuss different possibilities by which RNA polymerase directly or indirectly participates in R‐loop formation in Escherichia coli. Finally, our results also indicate that what determines the steady‐state level of hypernegatively supercoiled DNA in topA null mutants is likely to be complex and involves a multitude of factors, including the status of RNA polymerase, transcription–translation coupling, the cellular level of RNase HI, the status of DNA gyrase and the rate of relaxation of supercoiled DNA.

Isolation and nucleotide sequence of the hemA gene of Escherichia coli K12

SummaryThe hemA gene of Escherichia coli K12 was cloned by complementation of a hemA mutant of this organism. Subcloning of the initial 6.0 kb HindIII fragment allowed the isolation of a 1.5 kb NheI-AvaI fragment which retained the ability to complement the hemA mutant. DNA sequencing by the dideoxy chain terminator method of Sanger showed the presence of an open reading frame (ORF) of 1254 nucleotides, which ends 6 nucleotides beyond the AvaI site. Primer extension experiments showed the existence of a putative transcription initiation site for the hemA gene, located at position 130. A possible promoter sequence was identified upstream from this transcription initiation site, and its functional activity was confirmed by the use of the pK01 promoter-probe vector. Protein synthesis in an in vitro coupled transcription-translation system showed a 46 kDa protein, which corresponds to the mol. wt. of the hemA protein, as deduced from the nucleotide sequence of the gene. No homology was found between the amino acid sequence of the hemA protein of E. coli K12 and known sequences of other Δ-aminolevulinic acid synthases (Δ-ALAS), suggesting that this protein is different from other Δ-ALAS enzymes.

RNase HI overproduction is required for efficient full-length RNA synthesis in the absence of topoisomerase I in Escherichia coli

It has long been known that Escherichia coli cells deprived of topoisomerase I (topA null mutants) do not grow. Because mutations reducing DNA gyrase activity and, as a consequence, negative supercoiling, occur to compensate for the loss of topA function, it has been assumed that excessive negative supercoiling is somehow involved in the growth inhibition of topA null mutants. However, how excess negative supercoiling inhibits growth is still unknown. We have previously shown that the overproduction of RNase HI, an enzyme that degrades the RNA portion of an R‐loop, can partially compensate for the growth defects because of the absence of topoisomerase I. In this article, we have studied the effects of gyrase reactivation on the physiology of actively growing topA null cells. We found that growth immediately and almost completely ceases upon gyrase reactivation, unless RNase HI is overproduced. Northern blot analysis shows that the cells have a significantly reduced ability to accumulate full‐length mRNAs when RNase HI is not overproduced. Interestingly, similar phenotypes, although less severe, are also seen when bacterial cells lacking RNase HI activity are grown and treated in the same way. All together, our results suggest that excess negative supercoiling promotes the formation of R‐loops, which, in turn, inhibit RNA synthesis.

Characterization of a Leishmania donovani gene encoding a protein that closely resembles a type IB topoisomerase

In order to clone the gene encoding a type I DNA topoisomerase from Leishmania donovani, a PCR-amplified DNA fragment obtained with degenerate oligodeoxyribonucleotides was used to screen a genomic library from this parasite. An open reading frame of 1905 bases encoding a putative protein of 635 amino acid residues was isolated. A substantial part of the protein shares a significant degree of homology with the sequence of other known members of the IB topoisomerase family, in a highly conserved region of these enzymes termed the core domain. However, homology is completely lost after this conserved central core. Moreover, no conventional active tyrosine site could be identified. In fact, the protein expressed in Escherichia coli did not show any relaxation activity in vitro and was unable to complement a mutant deficient in topoisomerase I activity. The results of Southern blot experiments strongly suggested that the cloned gene was not a pseudogene. Northern analysis revealed that the gene was transcribed in its full length and also excluded the possibility that some form of splicing is necessary to produce a mature messenger. Furthermore, our results indicate that the gene is preferentially expressed in actively growing L.donovani promastigotes and that it is also expressed in other kinetoplastid parasites.

Isolation of the topB gene encoding DNA topoisomerase III as a multicopy suppressor of topA null mutations in Escherichia coli

One major function of DNA topoisomerase I in Escherichia coli is to repress R‐loop formation during transcription elongation, which may otherwise inhibit cell growth. We have previously shown that the growth problems of topA mutants can be corrected by overproducing RNase H, an enzyme that degrades the RNA moiety of an R‐loop. The goal of the present study was to identify other potential regulators of R‐loop formation. To this end, we have screened for multicopy suppressors of topA null mutations. As expected using this procedure, we cloned the rnhA gene encoding RNase H. In addition, we also identified the topB gene encoding DNA topoisomerase III as an efficient suppressor of topA null mutations and, hence, of R‐loop formation. We show that DNA topoisomerase III is able to relax transcription‐induced negative supercoiling both in vitro and in vivo. An R‐loop is also shown to be a hot‐spot for relaxation by DNA topoisomerase III, and we found that R‐loop‐dependent hypernegative supercoiling can be prevented by the activity of this topoisomerase in vivo. It is also shown that the topB gene can act synergistically with the rnhA gene to correct the growth defect of topA null mutants efficiently. This synergistic effect can be explained by the fact that some R‐loops must not be degraded in order for the RNA to be available for protein synthesis. Topoisomerase III can presumably repress the formation of such R‐loops or cause their destabilization to prevent RNA degradation. This is supported by the fact that overproduction of this topoisomerase corrects the negative effect of overexpressing RNase H activity on the growth of topA null mutants at low temperatures. Moreover, the fact that DNA topoisomerase III does not relax global supercoiling supports our previous conclusion that R‐loop formation, and therefore the essential function of DNA topoisomerase I, involves local, rather than global, supercoiling.