Mechanistic studies of transcription arrest at the adenovirus major late attenuation site. Comparison of purified RNA polymerase II and washed elongation complexes.
Transcription elongation in a nuclear extract in vitro is efficiently blocked by Sarkosyl at a specific site downstream of the adenovirus major late (ML) promoter at which regulated transcription arrest has also been observed in vivo. In the experiments reported here, we examined the response of the polymerase to the ML attenuation site in two assay systems: 1) purified RNA polymerase II (pol II) transcribing tailed templates and 2) elongation complexes formed on immobilized templates and then depleted of elongation factors by extensive washing. Efficient site-specific arrest occurred in both systems, demonstrating that recognition of the site is an intrinsic property of the polymerase. However, the elongation properties of washed elongation complexes and purified pol II were not equivalent. In particular, the efficiency of arrest of washed elongation complexes was influenced both by the promoter from which transcription was initiated and by DNA sequences upstream from the attenuation site that did not contribute to the arrest of purified pol II. The polymerase and washed elongation complexes both remained in stable ternary complexes at the ML site with a lifetime of hours; addition of the elongation factor SII to these complexes promoted resumption of elongation. The efficiency of arrest in both systems was dependent on the solution concentration of the nucleotide incorporated at +187 (just beyond the attenuation site), indicating that pausing is an important part of the arrest mechanism. Based on this and other findings, we argue that the polymerase assumes an altered, elongation-incompetent conformation when arrest occurs.
Wiest
D K
DK
Institute of Molecular Biology, University of Oregon, Eugene 97403.
Wang
D
D
Hawley
D K
DK
D000256:Adenoviridae / Q000235:genetics*; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D004279:DNA, Viral; D004268:DNA-Binding Proteins / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D010957:Plasmids; D011401:Promoter Regions, Genetic; D012319:RNA Polymerase II / Q000302:isolation & purification / Q000378:metabolism*; D012521:Sarcosine / Q000031:analogs & derivatives / Q000494:pharmacology; D013698:Templates, Genetic; D013728:Terminator Regions, Genetic; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic; D014764:Viral Proteins
D004279:DNA, Viral; D004268:DNA-Binding Proteins; C051096:MLTF protein, adenovirus; D014157:Transcription Factors; D014764:Viral Proteins; C025231:sarkosyl; D006160:Guanosine Triphosphate; D012319:RNA Polymerase II; D012521:Sarcosine