Transcription-dependent structural changes in the C-terminal domain of mammalian RNA polymerase subunit IIa/o.
The C-terminal domain of mammalian RNA polymerase subunit IIa consists of 52-tandem repeats of the consensus sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser. This C-terminal domain is essentially unmodified in RNA polymerase IIA and extensively phosphorylated in RNA polymerase IIO. A monoclonal antibody directed against the C-terminal domain was shown by kinetic enzyme-linked immunosorbent assay to have a 10-fold higher reactivity with RNA polymerase IIA than with RNA polymerase IIO. The ability of increasing concentrations of this monoclonal antibody to inhibit the initiation and elongation phase of transcription was determined. Although both phases of the transcription reaction were inhibited, a 10-fold higher concentration of antibody was required to inhibit elongation than was required to inhibit initiation. These results support the hypothesis that RNA polymerase IIA, containing an unphosphorylated C-terminal domain, is involved in the formation of an initiated complex, whereas elongation is catalyzed by RNA polymerase IIO, containing a phosphorylated C-terminal domain. Further indication that the C-terminal domain undergoes a structural change during the transcription cycle results from the observation that this domain is 3-fold more sensitive to clostripain cleavage in the elongation enzyme than in the free enzyme.
Laybourn
P J
PJ
Department of Biochemistry and Biophysics, University of California, Davis 95616.
Dahmus
M E
ME
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000915:Antibody Affinity; D000937:Antigen-Antibody Reactions; D002417:Cattle; D003546:Cysteine Endopeptidases / Q000378:metabolism; D066298:In Vitro Techniques; D046911:Macromolecular Substances; D010766:Phosphorylation; D011487:Protein Conformation; D012319:RNA Polymerase II / Q000378:metabolism*; D014158:Transcription, Genetic
D000911:Antibodies, Monoclonal; D046911:Macromolecular Substances; D012319:RNA Polymerase II; D003546:Cysteine Endopeptidases; C019913:clostripain