Sarkosyl defines three intermediate steps in transcription initiation by RNA polymerase III: application to stimulation of transcription by E1A.
We used Sarkosyl to analyze steps along the pathway of transcription initiation by RNA polymerase III. Sarkosyl (0.015%) inhibited transcription when present prior to incubation of RNA polymerase III, TFIIIB, and TFIIIC with the VAI gene, whereas it had no detectable effect on initiation or reinitiation of transcription when added subsequently. The formation of the corresponding 0.015% Sarkosyl-resistant complex required the presence of TFIIIC, TFIIIB, and RNA polymerase III but not nucleoside triphosphates. The addition of 0.05% Sarkosyl after this early step selectively inhibited a later step in the preinitiation pathway, allowing a single round of transcription after nucleoside triphosphate addition but blocking subsequent rounds of initiation. This step occurred prior to initiation because nucleoside triphosphates were not required for the formation of the corresponding 0.05% Sarkosyl-resistant complex. These observations provided a means to distinguish effects of regulatory factors on different steps in promoter activation and function. Using 0.05% Sarkosyl to limit reinitiation, we determined that the E1A-mediated stimulation of transcription by RNA polymerase III resulted from an increase in the number of active transcription complexes.
Kovelman
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Laboratory of Biochemistry and Molecular Biology, Rockefeller University, New York, New York 10021-6399.
Roeder
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RG
D017456:Adenovirus Early Proteins; D004268:DNA-Binding Proteins / Q000235:genetics; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D003902:Detergents; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D009856:Oncogene Proteins, Viral / Q000235:genetics*; D012320:RNA Polymerase III / Q000378:metabolism*; D012521:Sarcosine / Q000031:analogs & derivatives* / Q000494:pharmacology; D035361:Transcription Factor TFIIIB; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D019474:Transcription Factors, TFIII; D014158:Transcription, Genetic / Q000187:drug effects; D000098832:Transcription Factor TFIIIC
D017456:Adenovirus Early Proteins; D004268:DNA-Binding Proteins; D003902:Detergents; D009856:Oncogene Proteins, Viral; D035361:Transcription Factor TFIIIB; D014157:Transcription Factors; D019474:Transcription Factors, TFIII; D000098832:Transcription Factor TFIIIC; C025231:sarkosyl; D012321:DNA-Directed RNA Polymerases; D012320:RNA Polymerase III; D012521:Sarcosine