RNase H Overproduction Corrects a Defect at the Level of Transcription Elongation during rRNA Synthesis in the Absence of DNA Topoisomerase I in Escherichia coli

It has been suggested that the major function of DNA topoisomerase I in Escherichia coli is to suppress the formation of R-loops, which could inhibit growth. Although the currently available data suggest that the inhibitory effect of R-loops is exerted at the level of gene expression, this has never been demonstrated. In the present report, we show that rRNA synthesis is significantly impaired at the level of transcription elongation in a bacterial strain lacking DNA topoisomerase I. We found that this inhibition is due to transcriptional blocks. RNase H overproduction is also shown to considerably reduce the extent of such transcriptional blocks during rRNA synthesis. Moreover, one of these transcriptional blockage sites is located within a region where extensive R-loop formation was previously shown to occur on a plasmid DNA in the absence of DNA topoisomerase I. Together, these results allow us to propose that an important function of DNA topoisomerase I is to inhibit the formation of R-loops, which may otherwise translate into roadblocks for RNA polymerases. Our results also highlight the potential regulatory role of DNA supercoiling at the level of transcription elongation. It has been suggested that the major function of DNA topoisomerase I in Escherichia coli is to suppress the formation of R-loops, which could inhibit growth. Although the currently available data suggest that the inhibitory effect of R-loops is exerted at the level of gene expression, this has never been demonstrated. In the present report, we show that rRNA synthesis is significantly impaired at the level of transcription elongation in a bacterial strain lacking DNA topoisomerase I. We found that this inhibition is due to transcriptional blocks. RNase H overproduction is also shown to considerably reduce the extent of such transcriptional blocks during rRNA synthesis. Moreover, one of these transcriptional blockage sites is located within a region where extensive R-loop formation was previously shown to occur on a plasmid DNA in the absence of DNA topoisomerase I. Together, these results allow us to propose that an important function of DNA topoisomerase I is to inhibit the formation of R-loops, which may otherwise translate into roadblocks for RNA polymerases. Our results also highlight the potential regulatory role of DNA supercoiling at the level of transcription elongation. nucleotide(s) Escherichia coli DNA topoisomerase I, a member of the type IA family of topoisomerases, specifically relaxes negatively supercoiled DNA (1.Wang J.C. J. Mol. Biol. 1971; 55: 523-533Crossref PubMed Scopus (530) Google Scholar, 2.Wang J.C. Annu. Rev. Biochem. 1996; 65: 635-692Crossref PubMed Scopus (2097) Google Scholar). This specificity is explained by the fact that this enzyme binds to the junction of single-stranded and double-stranded DNA regions. DNA opening, and hence the generation of single-stranded DNA regions, is promoted by negative but not positive supercoiling. Hot spots for relaxation by DNA topoisomerase I are provided during transcription elongation in the frame of the twin-domain model (3.Liu L.F. Wang J.C. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 7024-7027Crossref PubMed Scopus (1576) Google Scholar). Indeed, very high levels of negative supercoiling can be generated behind the moving RNA polymerase during transcription elongation (4.Wu H.Y. Shyy S.H. Wang J.C. Liu L.F. Cell. 1988; 53: 433-440Abstract Full Text PDF PubMed Scopus (579) Google Scholar, 5.Tsao Y.P. Wu H.-Y. Liu L.F. Cell. 1989; 56: 111-118Abstract Full Text PDF PubMed Scopus (285) Google Scholar). An R-loop, in which the template strand is paired with the nascent RNA, leaving the nontemplate strand unpaired, also provides a hot spot for relaxation by this enzyme (6.Phoenix P. Raymond M.-A. Massé E. Drolet M. J. Biol. Chem. 1997; 272: 1473-1479Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). The accumulated evidence over the last few years has allowed us to conclude that a major function of DNA topoisomerase I in E. coli is to inhibit R-loop formation during transcription elongation. Indeed, the growth problem of topA (encoding DNA topoisomerase I) null mutants was shown to be partially corrected by overproducing RNase H, an enzyme that degrades the RNA moiety of an R-loop (7.Drolet M. Phoenix P. Menzel R. Massé E. Liu L.F. Crouch R.J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3526-3530Crossref PubMed Scopus (206) Google Scholar). A correlation was also established between the level of DNA gyrase activity, the enzyme that introduces negative supercoiling within the chromosomal DNA, and the amount of RNase H required to stimulate the growth of topA null mutants (7.Drolet M. Phoenix P. Menzel R. Massé E. Liu L.F. Crouch R.J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3526-3530Crossref PubMed Scopus (206) Google Scholar) and to inhibit R-loop formation during transcription (8.Massé E. Drolet M. J. Biol. Chem. 1999; 274: 16659-16664Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). The finding that severaltopA null mutants carry compensatory gyrmutations (in gyrA or gyrB) that reduce DNA gyrase activity and correct their growth defect (9.DiNardo S. Voelkel K.A. Sternglanz R. Reynolds A.E. Wright A. Cell. 1982; 31: 43-51Abstract Full Text PDF PubMed Scopus (295) Google Scholar, 10.Pruss G.J. Manes S.H. Drlica K. Cell. 1982; 31: 35-42Abstract Full Text PDF PubMed Scopus (243) Google Scholar) was therefore explained by the supercoiling activity of DNA gyrase, which promotes R-loop formation (7.Drolet M. Phoenix P. Menzel R. Massé E. Liu L.F. Crouch R.J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3526-3530Crossref PubMed Scopus (206) Google Scholar). On the contrary, DNA topoisomerase I activity inhibits R-loop formation. The results of in vitroexperiments very well support this model in which DNA topoisomerases with opposing enzymatic activities control the formation of growth-inhibitory R-loops (6.Phoenix P. Raymond M.-A. Massé E. Drolet M. J. Biol. Chem. 1997; 272: 1473-1479Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 11.Drolet M. Bi X. Liu L.F. J. Biol. Chem. 1994; 269: 2068-2074Abstract Full Text PDF PubMed Google Scholar, 12.Massé E. Phoenix P. Drolet M. J. Biol. Chem. 1997; 272: 12816-12823Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). More recent data have suggested that local supercoiling generated during transcription, rather than global supercoiling, which reflects the average superhelical density of all supercoiling domains, is responsible for R-loop formation and hence is linked to the essential function of DNA topoisomerase I (8.Massé E. Drolet M. J. Biol. Chem. 1999; 274: 16659-16664Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 13.Massé E. Drolet M. J. Biol. Chem. 1999; 274: 16654-16658Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). The mechanism(s) by which R-loop formation exerts its growth inhibitory effects is still unknown. The fact that topA null mutants are sensitive to changes in environmental conditions (7.Drolet M. Phoenix P. Menzel R. Massé E. Liu L.F. Crouch R.J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3526-3530Crossref PubMed Scopus (206) Google Scholar, 14.Dorman C.J. Lynch A.S. Ni Bhriain N. Higgins C.F. Mol. Microbiol. 1989; 3: 531-540Crossref PubMed Scopus (48) Google Scholar, 15.Qi H. Menzel R. Tse-Dinh Y.C. Mol. Microbiol. 1996; 21: 703-711Crossref PubMed Scopus (29) Google Scholar, 16.Qi H. Menzel R. Tse-Dinh Y.C. J. Mol. Biol. 1997; 267: 481-489Crossref PubMed Scopus (38) Google Scholar, 17.Massé E. Drolet M. J. Mol. Biol. 1999; 294: 321-332Crossref PubMed Scopus (45) Google Scholar) may suggest that gene expression is somehow impaired by R-loop formation. For example, the finding that RNase H overproduction allowstopA null mutants to more rapidly adapt to fresh media and to nutritional shift-ups, may suggest that R-loops inhibit the expression of genes required for such growth transitions (7.Drolet M. Phoenix P. Menzel R. Massé E. Liu L.F. Crouch R.J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3526-3530Crossref PubMed Scopus (206) Google Scholar, 17.Massé E. Drolet M. J. Mol. Biol. 1999; 294: 321-332Crossref PubMed Scopus (45) Google Scholar). Interestingly, we have shown previously that R-loop formation can occur during transcription of a DNA fragment carrying a portion of therrnB operon on a plasmid DNA, in the absence of DNA topoisomerase I (12.Massé E. Phoenix P. Drolet M. J. Biol. Chem. 1997; 272: 12816-12823Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). It has also been shown that it is during nutritional shift-up conditions that E. coli cells have the highest requirement for rRNA synthesis (encoded byrrn operons; Ref. 18.Condon C. Liveris D. Squires C. Schwartz I. Squires C.L. J. Bacteriol. 1995; 177: 4152-4156Crossref PubMed Scopus (171) Google Scholar). In this report, we present data suggesting that R-loop formation inhibits rRNA synthesis at the level of transcription elongation. Our results allow us to propose that DNA topoisomerase I can act as a transcription elongation factor that inhibits R-loop-dependent transcriptional blocks. These findings can have an important impact on our understanding of the mechanism(s) by which DNA topoisomerases and DNA supercoiling influence gene expression. E. coli strains used are listed in Table I. Details of their construction by transduction using P1vir phage are also provided in Table I. pSK760 is a pBR322 derivative carrying thernhA gene encoding RNase H (22.Kanaya S. Crouch R.J. J. Biol. Chem. 1983; 258: 1276-1281Abstract Full Text PDF PubMed Google Scholar).Table IE. coli strains used in this studyStrainGenotypeRef./constructionAQ634ilv, metB, his-29, trpA9605, pro, thyA, deoB (or C)19.Masai H. Asai T. Kubota Y. Arai K. Kogoma T. EMBO J. 1994; 13: 5338-5345Crossref PubMed Scopus (93) Google ScholarCAG18592zie-3163::Tn10kan20.Singer M. Baker T.A. Schnitzler G. Deischel S.M. Goel M. Dove W. Jaacks K.J. Grossman A.D. Erickson J.W. Gross C.A. Microbiol. Rev. 1989; 53: 1-24Crossref PubMed Google ScholarRFM445gyrB221(couR)gyrB203(Ts)7.Drolet M. Phoenix P. Menzel R. Massé E. Liu L.F. Crouch R.J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3526-3530Crossref PubMed Scopus (206) Google ScholarPH326RFM445zie-3163::Tn10kanRFM445XP1.CAG18592, select Kmr and TsMA249AQ634gyrB221(couR) gyrB203(Ts)zie-3163::Tn10kanAQ634XP1.PH326, select Kmr and TsRFM480topA20::Tn107.Drolet M. Phoenix P. Menzel R. Massé E. Liu L.F. Crouch R.J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3526-3530Crossref PubMed Scopus (206) Google ScholarMA251MA249topA20::Tn10MA249XP1.RFM480, select Tetr and cold sensitivity (27 °C)K37garB10, fhuA22, phoA4, ompF627, serU132, fadL701, relA1, pit-10, spoT1, rrnB-2, mcrB1, creC51021.Friedman D.I. Baumann M. Baron L.S. Virology. 1976; 73: 119-127Crossref PubMed Scopus (66) Google ScholarK450K37nusB521.Friedman D.I. Baumann M. Baron L.S. Virology. 1976; 73: 119-127Crossref PubMed Scopus (66) Google Scholar Open table in a new tab Unless otherwise indicated, the strains in (7.Drolet M. Phoenix P. Menzel R. Massé E. Liu L.F. Crouch R.J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3526-3530Crossref PubMed Scopus (206) Google Scholar) with the required indicated, at was to to and to in fresh with cold at and to an A of to and for the in the to and was by of at to of RNA was of and of by a of the of H. S. J. Biol. Chem. Full Text PDF PubMed Google Scholar). of by the cells with the the at the required amount to the used in the The RNA was in of and at of the RNA was by in as J. T. A Scholar). the was In to the the for with and to of the RNA was used for by using at to J. T. A Scholar). was used in all the it the of RNA at the of all E. coli The of the and on RNA as on J. T. A Scholar). the RNA was to a and to J. T. A Scholar). The a portion of the was by polymerase using DNA S. M. Cell. 1983; Full Text PDF PubMed Scopus Google Scholar) and with the and the to J. T. A Scholar) and to the impact of the of DNA topoisomerase I activity on rRNA strains carrying a of these also and is therefore of topoisomerase I The of these was used previously to the between R-loop formation and growth inhibition in the absence of DNA topoisomerase I (7.Drolet M. Phoenix P. Menzel R. Massé E. Liu L.F. Crouch R.J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3526-3530Crossref PubMed Scopus (206) Google Scholar). Indeed, such a topA null is to at DNA gyrase activity is to for the absence of DNA topoisomerase I, therefore R-loop formation to a level for the the DNA gyrase more in R-loop formation (8.Massé E. Drolet M. J. Biol. Chem. 1999; 274: 16659-16664Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). RNase H overproduction was shown to significantly the growth of null to (7.Drolet M. Phoenix P. Menzel R. Massé E. Liu L.F. Crouch R.J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3526-3530Crossref PubMed Scopus (206) Google Scholar). the synthesis of rRNA and RNA was for of with could at the level of transcription at the level of rRNA The of the rRNA to the rRNA for a of an in these could also during transcription elongation in the of and rRNA The of RNA of cells at in rRNA synthesis in the of the to between and not The of was with cells to a for the growth of In to show that our can be used to at the level of transcription we a of and The and was shown previously to be in of rRNA transcription M. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). of such a defect is an in rRNA transcription and in the of rRNA synthesis M. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). for and In our shown to be to a amount of not This that in the of rRNA a be to rRNA but rather be to in rRNA synthesis and cells at to of at which to for with for and shown previously M. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google our results that the rRNA synthesis is in as with the in the strain rRNA and of rRNA to Our is therefore to in rRNA synthesis at the level of transcription elongation. in also show that the rRNA synthesis is significantly null is to and that the topA null at rRNA a with the strain not It can also be that the of to rRNA synthesis is significantly in the topA null and also that RNase H overproduction in the topA null the rRNA synthesis and and the to and These results suggest that R-loop formation during rRNA synthesis a defect at the level of transcription or elongation at the level of rRNA with an to the rRNA P. S. M. Cell. 1983; Full Text PDF PubMed Scopus Google Scholar, R. S. N. G. J. Mol. Biol. 1994; PubMed Scopus Google Scholar, M. H. G. Microbiol. 1996; PubMed Google Scholar) that the in the rRNA synthesis in null be to a in transcription In results in may suggest that transcription is in the topA null not overproducing RNase H and shown M. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google it can also be that rRNA transcription is in the and In with the negative model for the of rRNA rRNA transcription is more to for a defect at the level of transcription elongation in the M. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, T. W. Annu. Rev. Microbiol. 1996; PubMed Scopus Google Scholar). A could also be to the results with null This is in the synthesis in a at a at to an A of as to and for and or and RNA was at the for the and as of the RNA was by in as of the its for the of to The by and by into the of within and The to rRNA for reflects very well the in this an was for the strain not The results shown are of to the at the of the RNA of the shown in was used for with a to the as The results shown are of Our the synthesis of In E. coli all of which are very are required to the rRNA into rRNA D. Annu. Rev. Microbiol. PubMed Scopus Google Scholar). In of rRNA is present as A in the rRNA synthesis could be explained by a defect in one or more of the required for rRNA this rRNA be which is not the for the topA null data not The in the is the of the rRNA that the rRNA has been This is by the formation of an a and a portion of the rRNA by RNase and the rRNA and the rRNA are The rRNA is rapidly and the rRNA is in to for or for transcriptional blocks RNA it was to a DNA more than the rRNA In this not rRNA or the rRNA could be by of RNA for the shown in with a DNA fragment a portion of the rRNA in a shown in the of at of the RNA with to the rRNA The amount of this RNA in strain very well with the of the by using an to a portion of the rRNA region The RNA in may rRNA that was This rRNA is in the that was previously shown to be in transcription during rRNA synthesis M. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). to and in in RNA topA null of the level of RNase H activity and These RNA also in the mutants at not Interestingly, the of the one is within a region where extensive R-loop formation was previously shown to occur transcription on a plasmid DNA in the absence of DNA topoisomerase I (12.Massé E. Phoenix P. Drolet M. J. Biol. Chem. 1997; 272: 12816-12823Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). These or transcriptional blocks Our with RNA mutants not overproducing RNase H that the rRNA is the RNA is cells for of to not This can be as an that one or are with This could transcriptional blocks with the RNA within RNA rather than with that are rapidly Moreover, a is with RNA cells to for of it can be that the of the is Indeed, it is that of the cells to the are the and therefore the results also with a bacterial strain in which the topA gene is Ref. M. Phoenix P. Menzel R. Massé E. Liu L.F. Crouch R.J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3526-3530Crossref PubMed Scopus (206) Google data not It is also that this is more the topA null not RNase H. This may that transcriptional blocks are with the level of RNase H activity is The results of suggest that this is between transcriptional blocks and we the of the RNA carrying the rRNA RNA was by the transcription to cells previously to for and by RNA the of the of carrying the rRNA a the of one of these of the that the RNA carrying the rRNA in the topA is very its than as previously shown for the rRNA and data not P. S. M. Cell. 1983; Full Text PDF PubMed Scopus Google Scholar, R. S. N. G. J. Mol. Biol. 1994; PubMed Scopus Google Scholar, M. H. G. Microbiol. 1996; PubMed Google Scholar). For this the reflects the it is the rRNA by for the null that the RNA carrying the rRNA region is very In we have found that the is still very of not The shown in A that the RNA has its within the rRNA region and the have their or the the very high of these the one with the within the rRNA we have to conclude that transcriptional blocks rather than are in transcriptional overproducing RNase H these and of the of RNA that the RNA with the within the rRNA region is at more in the RNase H is not For the and the RNase H is not these may be by the fact that RNA polymerase may sites to more with The fact that the is more than the more may the absence of a of RNA polymerase of in the The results in this fact and the high of the transcriptional blocks null mutants RNase H is not Indeed, such still of to to the of of the RNA at at to an A of as and to of at this at was to the and the RNA was at the the RNA was the of of the RNA used for with a to the as The in A the and the is an of the In the of the for the RNA was by to the of of these RNA The results shown are of The is and the is to the very high of the RNA at in topA null at to of as and to of at this at was to the and the RNA was at the the RNA was the of of the RNA used for with a to the as The the and the is an of the Although it has been for a that are to growth and that DNA supercoiling is somehow in this inhibition (9.DiNardo S. Voelkel K.A. Sternglanz R. Reynolds A.E. Wright A. Cell. 1982; 31: 43-51Abstract Full Text PDF PubMed Scopus (295) Google Scholar, 10.Pruss G.J. Manes S.H. Drlica K. Cell. 1982; 31: 35-42Abstract Full Text PDF PubMed Scopus (243) Google Scholar, K. Mol. Microbiol. PubMed Scopus Google mechanism(s) has been to such a negative of the that R-loop formation could somehow be in this effect the results of in in which extensive shown to occur during transcription in the of DNA gyrase and in the absence of DNA topoisomerase I M. Bi X. Liu L.F. J. Biol. Chem. 1994; 269: 2068-2074Abstract Full Text PDF PubMed Google Scholar). A finding was the that RNase H overproduction can partially the growth defect null mutants (7.Drolet M. Phoenix P. Menzel R. Massé E. Liu L.F. Crouch R.J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3526-3530Crossref PubMed Scopus (206) Google Scholar). It was that R-loops could act by or such as is suggested to occur in T. Microbiol. Mol. Biol. Rev. 1997; PubMed Scopus Google or by transcription elongation. In the present we have shown that transcriptional blocks in the absence of DNA topoisomerase I can be by overproducing RNase H. Interestingly, we previously found that RNase H the RNA synthesis in a negatively supercoiled template was (6.Phoenix P. Raymond M.-A. Massé E. Drolet M. J. Biol. Chem. 1997; 272: 1473-1479Abstract Full Text Full Text PDF PubMed Scopus (52) Google E. Phoenix P. Drolet M. J. Biol. Chem. 1997; 272: 12816-12823Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). Massé and M. Moreover, one of the transcriptional blocks in the present was found within a region where extensive R-loop formation was during in and in transcription on plasmid in the absence of DNA topoisomerase I (6.Phoenix P. Raymond M.-A. Massé E. Drolet M. J. Biol. Chem. 1997; 272: 1473-1479Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 12.Massé E. Phoenix P. Drolet M. J. Biol. Chem. 1997; 272: 12816-12823Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). Together, these allow us to propose that the R-loops generated due to the absence of DNA topoisomerase I roadblocks for RNA polymerases. In our the nascent RNA the template DNA strand behind a moving RNA to an R-loop This R-loop a for the RNA For the we can on the effects of such transcriptional blocks are on the growth null RNase H overproduction their growth at (7.Drolet M. Phoenix P. Menzel R. Massé E. Liu L.F. Crouch R.J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3526-3530Crossref PubMed Scopus (206) Google Scholar). Moreover, our not allow to between the of E. we a the of of these in transcriptional The fact that a topA null at rRNA than an strain during a us to that the transcriptional blocks to the growth in the absence of DNA topoisomerase I. This is also by our finding that the growth of a topA null overproducing RNase H at can be by the of Drolet and C. This is by a plasmid carrying a operon within the topA null The transcriptional blocks within to be very This is suggested by the results of and with RNA cells to Indeed, a was still by with a DNA fragment to the rRNA region an of to This high is also by the fact that the transcriptional sites the region of the topA null cells to This is the of transcriptional blocks in the region transcriptional activity, and transcriptional blockage in this Moreover, our show that the of the RNA carrying the rRNA with the of of the topA null to not This also that transcriptional blocks are with could such a high an R-loop is in transcriptional such a high may the of RNase H. It is also that the R-loop, behind the moving RNA provides a single-stranded DNA to which or single-stranded can this may with the moving RNA RNase H not act rapidly a very and DNA region may a could the of the RNA The formation of RNase may also be For example, transcription of a was shown to the formation of a very that the of an and that inhibits transcription elongation E. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar). It also be to the RNase can inhibit as a the formation of double-stranded M. EMBO J. 1997; PubMed Scopus Google Scholar). In we previously that the of can be very high in topA null In of the results in this and the effects of topA K. Mol. Microbiol. PubMed Scopus Google Scholar, K. Microbiol. Rev. PubMed Google Scholar, C.J. Ni Bhriain N. Microbiol. Full Text PDF PubMed Scopus Google we the that transcription elongation be more than previously in the control of gene expression. our has been one that the effect of DNA supercoiling on transcription elongation in M. R. Mol. Microbiol. PubMed Scopus Google Scholar). In that in transcriptional within the region of the operon shown to with the level of negative supercoiling of the DNA Moreover, new sites at negative superhelical of the DNA a can be established between these and the results in the present to be demonstrated. In we that be the effects of DNA supercoiling and DNA topoisomerases on gene expression to transcription More may effects at the level of transcription elongation rather than We D. I. for the of bacterial strains and for of the

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