Use of a novel S1 nuclease RNA-mapping technique to measure efficiency of transcription termination on polyomavirus DNA.
We devised a strategy to measure the efficiency of transcription termination in vivo by RNA polymerase on polyomavirus DNA. Pulse-labeled nuclear RNA was hybridized with a single-stranded polyomavirus DNA fragment which spans the transcription initiation region. Hybrids were treated with RNase, bound to nitrocellulose filters, eluted with S1 nuclease, and analyzed by gel electrophoresis. The ratio of full-length to less-than-full-length DNA-RNA hybrids was used to calculate transcription termination frequency. We found that 50% of the polymerases terminated per traverse of the L DNA strand during the late phase of infection. The method for mapping in vivo pulse-labeled RNAs which we developed is potentially useful for studying unstable cellular or viral RNAs.
D000818:Animals; D004279:DNA, Viral / Q000235:genetics*; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D004720:Endonucleases; D051379:Mice; D009693:Nucleic Acid Hybridization; D011120:Polyomavirus / Q000235:genetics*; D012367:RNA, Viral / Q000235:genetics*; D015719:Single-Strand Specific DNA and RNA Endonucleases; D014158:Transcription, Genetic
D004279:DNA, Viral; D012367:RNA, Viral; D012321:DNA-Directed RNA Polymerases; D004720:Endonucleases; D015719:Single-Strand Specific DNA and RNA Endonucleases