Effect of varying the supercoiling of DNA on transcription and its regulation.
The effect of superhelicity of DNA templates on transcription is well documented in several cases. However, the amount of supercoiling that is needed to bring about any changes and the steps at which such effects are exerted were not systematically studied. We investigated the effect of DNA supercoiling on transcription from a set of promoters present on a plasmid by using a series of topoisomers with different superhelical densities ranging from totally relaxed to more than physiological. In vitro transcription assays with these topoisomers in the absence and presence of gene regulatory proteins showed that the effect of negative supercoiling on intrinsic transcription varies from promoter to promoter. Some of those promoters, in which DNA superhelicity stimulated transcription, displayed specific optima of superhelical density while others did not. The results also showed that the amounts of abortive RNA synthesis from two of the promoters decreased and full-length RNA increased with increasing supercoiling, indicating for the first time an inverse relationship between full-length and abortive RNA synthesis and supporting a role of DNA superhelicity in promoter clearance. DNA supercoiling might also influence the point of RNA chain termination. Furthermore, the effect of varying the amount of supercoiling on the action of gene regulatory proteins suggested the mode of action, which is consistent with previous results. Our results underscore the importance of DNA supercoiling in fine-tuning promoter activities, which should be relevant in cell physiology given that local changes in chromosomal supercoiling must occur in different environments.
Lim
Heon M
HM
Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892-4264, USA.
Lewis
Dale E A
DE
Lee
Hee Jung
HJ
Liu
Mofang
M
Adhya
Sankar
S
D000818:Animals; D002417:Cattle; D004264:DNA Topoisomerases, Type I / Q000378:metabolism; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism; D004278:DNA, Superhelical / Q000235:genetics* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial / Q000235:genetics; D007763:Lac Operon / Q000235:genetics; D009690:Nucleic Acid Conformation; D010957:Plasmids / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics; D012329:RNA, Bacterial / Q000032:analysis / Q000096:biosynthesis; D014158:Transcription, Genetic
D004269:DNA, Bacterial; D004278:DNA, Superhelical; D012329:RNA, Bacterial; D004264:DNA Topoisomerases, Type I