Dynamics of DNA supercoiling by transcription in Escherichia coli.
The relative rotation between RNA polymerase and DNA during transcription elongation can lead to supercoiling of the DNA template. However, the variables that influence the efficiency of supercoiling by RNA polymerase in vivo are poorly understood, despite the importance of supercoiling for DNA metabolism. We describe a model system to measure the rate of supercoiling by transcription and to estimate the rates of topoisomerase turnover in Escherichia coli. Transcription in a strain lacking topoisomerase I can lead to optimal supercoiling, wherein nearly one positive and one negative superturn are produced for each 10.4 base pairs transcribed. This rapid efficient supercoiling is observed during transcription of membrane-associated gene products, encoded by tet (the gene for tetracycline resistance) and phoA (the gene for E. coli alkaline phosphatase), when the genes are oppositely oriented. Replacement of tet by cat, the gene from Tn9 encoding resistance to chloramphenicol, whose gene product is soluble in the cytosol, reduces the efficiency of supercoiling by RNA polymerase. In a wild-type topoisomerase background, both gyrase and topoisomerase I are kinetically competent to relieve superturns produced by transcription. These results suggest that the level of DNA supercoiling in vivo is probably determined by topoisomerase activity, not by transcription.
Cook
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Melvin Calvin Laboratory, Department of Chemistry, University of California, Berkeley.
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Pon
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Hearst
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D000469:Alkaline Phosphatase / Q000235:genetics; D001483:Base Sequence; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D004264:DNA Topoisomerases, Type I / Q000378:metabolism; D004250:DNA Topoisomerases, Type II / Q000378:metabolism; D004251:DNA Transposable Elements; D004278:DNA, Superhelical / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D005798:Genes, Bacterial; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D010957:Plasmids; D013753:Tetracycline Resistance / Q000235:genetics; D014158:Transcription, Genetic
D004251:DNA Transposable Elements; D004278:DNA, Superhelical; D009838:Oligodeoxyribonucleotides; D015500:Chloramphenicol O-Acetyltransferase; D012321:DNA-Directed RNA Polymerases; D000469:Alkaline Phosphatase; D004264:DNA Topoisomerases, Type I; D004250:DNA Topoisomerases, Type II