C-terminal domain phosphatase sensitivity of RNA polymerase II in early elongation complexes on the HIV-1 and adenovirus 2 major late templates.
The fate of RNA polymerase II in early elongation complexes is under the control of factors that regulate and respond to the phosphorylation state of the C-terminal domain (CTD). Phosphorylation of the CTD protects early elongation complexes from negative transcription elongation factors such as NELF, DSIF, and factor 2. To understand the relationship between transcript elongation and the sensitivity of RNA polymerase IIO to dephosphorylation, elongation complexes at defined positions on the Ad2-ML and human immunodeficiency virus type 1 (HIV-1) templates were purified, and their sensitivity to CTD phosphatase was determined. Purified elongation complexes treated with 1% Sarkosyl and paused at U(14)/G(16) on an HIV-1 template and at G(11) on the Ad2-ML template are equally sensitive to dephosphorylation by CTD phosphatase. Multiple elongation complexes paused at more promoter distal sites are more resistant to dephosphorylation than are U(14)/G(16) and G(11) complexes. The HIV-1 long terminal repeat and adenovirus 2 major late promoter do not appear to differentially influence the CTD phosphatase sensitivity of stringently washed complexes. Subsequent elongation by 1% Sarkosyl-washed U(14)/G(16) complexes is unaffected by prior CTD phosphatase treatment. This result is consistent with the hypothesis that CTD phosphatase requires the presence of specific elongation factors to propagate a negative effect on transcript elongation. The action of CTD phosphatase on elongation complexes is inhibited by HIV-1 Tat protein. This observation is consistent with the idea that Tat suppression of CTD phosphatase plays a role in transactivation.
Section of Molecular and Cellular Biology, Division of Biological Sciences, University of California, Davis, California 95616, USA.
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Marshall
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Dahmus
D000260:Adenoviruses, Human / Q000235:genetics*; D001483:Base Sequence; D002467:Cell Nucleus / Q000378:metabolism; D003902:Detergents; D016325:HIV Long Terminal Repeat; D015497:HIV-1 / Q000235:genetics*; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D012319:RNA Polymerase II / Q000378:metabolism*; D012521:Sarcosine / Q000031:analogs & derivatives / Q000494:pharmacology; D013698:Templates, Genetic; D014158:Transcription, Genetic
D003902:Detergents; C025231:sarkosyl; D012319:RNA Polymerase II; D010749:Phosphoprotein Phosphatases; C116022:carboxy-terminal domain phosphatase; D012521:Sarcosine