Cook D N, Ma D, Pon N G, Hearst J E
Melvin Calvin Laboratory, Department of Chemistry, University of California, Berkeley.
Proc Natl Acad Sci U S A. 1992 Nov 15;89(22):10603-7. doi: 10.1073/pnas.89.22.10603.
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.
转录延伸过程中RNA聚合酶与DNA之间的相对旋转会导致DNA模板超螺旋化。然而，尽管超螺旋化对DNA代谢很重要，但影响体内RNA聚合酶超螺旋化效率的变量却知之甚少。我们描述了一个模型系统，用于测量转录产生超螺旋的速率，并估计大肠杆菌中拓扑异构酶周转的速率。在缺乏拓扑异构酶I的菌株中进行转录可导致最佳超螺旋化，即每转录10.4个碱基对会产生近一个正超螺旋和一个负超螺旋。当由tet（四环素抗性基因）和phoA（大肠杆菌碱性磷酸酶基因）编码的膜相关基因产物的基因呈相反方向排列时，在转录过程中会观察到这种快速有效的超螺旋化。用cat取代tet，cat是来自Tn9的编码氯霉素抗性的基因，其基因产物可溶于细胞质，这会降低RNA聚合酶超螺旋化的效率。在野生型拓扑异构酶背景下，gyrase和拓扑异构酶I在动力学上都有能力缓解转录产生的超螺旋。这些结果表明，体内DNA超螺旋化水平可能由拓扑异构酶活性决定，而非转录。
