Unwinding of the DNA helix in simian virus 40 chromosome templates by RNA polymerase

Abstract

We measured the distortion of the DNA helix by RNA polymerase transcribing simian virus 40 (SV40) chromosome templates and compared it with the distortion caused by the enzymes as it transcribes naked SV40 DNA, using RNA polymerase from Escherichia coli. Purified DNA topoisomerase I was added to the transcription reactions and the number of supercoil turns in DNA, after deproteinising and removal of RNA, was determined by gel electrophoresis and band-counting. The number of polymerase molecules bound per naked DNA molecule was determined by electron microscopy. Each bound RNA polymerase distorted the template in such a way as to lead to on apparent average of 0.6-0.7 negative superhelical turn in the extracted DNA. Thus only few base-pairs are melted per RNA polymerase molecule. When SV40 chromosomes were transcribed the extracted DNA had a higher number of supercoil turns than DNA extracted from the initial chromosomes. We conclude that the polymerase deforms the DNA in chromatin in the same way as it deforms pure DNA. From control experiments with inhibitors of initiation we estimated that on average 9-10 RNA polymerase molecules were bound per SV40 chromosome. This suggests that transcription can proceed while the majority or all the nucleosomal structures are intact on an SV40 DNA molecule. We discuss the implications of these findings for the mechanism of transcription of chromatin.