Brabant F, Acheson N H
Department of Microbiology and Immunology, McGill University, Montreal, Quebec, Canada.
J Virol. 1995 Jul;69(7):4423-30. doi: 10.1128/JVI.69.7.4423-4430.1995.
RNA polymerase II molecules that transcribe the late strand of the 5.3-kb circular polyomavirus genome stall just upstream of the DNA replication origin, in a region containing multiple binding sites for polyomavirus large T antigen. Stalling of RNA polymerases depends on the presence of functional large T antigen and on the integrity of large T antigen binding site A. To gain insight into the interaction between DNA-bound large T antigen and RNA polymerase II, we mapped the position of stalled RNA polymerases by analyzing nascent RNA chains associated with these polymerases. Elongation of RNA in vitro, followed by hybridization with a nested set of DNA fragments extending progressively farther into the stalling region, allowed localization of the 3' end of the nascent RNA to a position 5 to 10 nucleotides upstream of binding site A. Ribonuclease treatment of nascent RNAs on viral transcription complexes, followed by in vitro elongation and hybridization, allowed localization of the distal end of stalled RNA polymerases to a position 40 nucleotides upstream of binding site A. This RNA footprint shows that elongating RNA polymerases stall at a site very close to the position of DNA-bound large T antigen and that they protect approximately 30 nucleotides of nascent RNA against ribonuclease digestion.
转录5.3kb环状多瘤病毒基因组后随链的RNA聚合酶II分子在DNA复制起点上游停滞，该区域含有多个多瘤病毒大T抗原的结合位点。RNA聚合酶的停滞取决于功能性大T抗原的存在以及大T抗原结合位点A的完整性。为深入了解与DNA结合的大T抗原和RNA聚合酶II之间的相互作用，我们通过分析与这些聚合酶相关的新生RNA链来确定停滞RNA聚合酶的位置。在体外进行RNA延伸，然后与一组嵌套的DNA片段杂交，这些片段逐渐延伸到停滞区域，从而将新生RNA的3'端定位到结合位点A上游5至10个核苷酸的位置。对病毒转录复合物上的新生RNA进行核糖核酸酶处理，然后进行体外延伸和杂交，可将停滞RNA聚合酶的远端定位到结合位点A上游40个核苷酸的位置。这种RNA足迹表明，正在延伸的RNA聚合酶在一个非常接近与DNA结合的大T抗原位置的位点停滞，并且它们保护大约30个核苷酸的新生RNA不被核糖核酸酶消化。
