Large-scale effects of transcriptional DNA supercoiling in vivo

The scale of negative DNA supercoiling generated by transcription in Top(+) Escherichia coli cells was assessed from the efficiency of cruciform formation upstream of a regulated promoter. An increase in negative supercoiling upon promoter induction led to cruciform formation, which was quantitative...

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Bibliographic Details
Published in:Journal of molecular biology 1999-10, Vol.292 (5), p.1149-1160
Main Authors: Krasilnikov, A S, Podtelezhnikov, A, Vologodskii, A, Mirkin, S M
Format: Article
Language:English
Subjects:
Chloramphenicol - pharmacology
Diffusion
DNA Topoisomerases, Type I - metabolism
DNA, Recombinant - genetics
DNA, Superhelical - chemistry
DNA, Superhelical - genetics
DNA, Superhelical - metabolism
DNA-Binding Proteins - genetics
DNA-Binding Proteins - metabolism
Escherichia coli
Escherichia coli - drug effects
Escherichia coli - enzymology
Escherichia coli - genetics
Escherichia coli - metabolism
Genes, Bacterial - genetics
Isomerism
Isopropyl Thiogalactoside - pharmacology
Nucleic Acid Conformation - drug effects
Plasmids - chemistry
Plasmids - genetics
Plasmids - metabolism
Promoter Regions, Genetic - genetics
Protein Binding
Repressor Proteins - genetics
Repressor Proteins - metabolism
Transcription, Genetic - drug effects
Transcription, Genetic - genetics
Viral Proteins
Viral Regulatory and Accessory Proteins
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Summary:The scale of negative DNA supercoiling generated by transcription in Top(+) Escherichia coli cells was assessed from the efficiency of cruciform formation upstream of a regulated promoter. An increase in negative supercoiling upon promoter induction led to cruciform formation, which was quantitatively measured by chemical probing of intracellular DNA. By placing a cruciform-forming sequence at varying distances from the promoter, we found that the half-dissociation length of transcription supercoiling wave is approximately 800 bp. This is the first proof that transcription can affect DNA structure on such a remarkably large scale in vivo. Moreover, cooperative binding of the cI repressor to the upstream promoter DNA did not preclude efficient diffusion of transcriptional supercoiling. Finally, our plasmids appeared to contain discrete domains of DNA supercoiling, defined by the features and relative orientation of different promoters.
ISSN:0022-2836
DOI:10.1006/jmbi.1999.3117