Phosphorylation of RNA polymerase IIA occurs subsequent to interaction with the promoter and before the initiation of transcription.
The largest subunit of mammalian RNA polymerase II contains at its C terminus an unusual domain consisting of multiple tandem repeats of the seven-amino acid consensus sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser. This domain is unphosphorylated in RNA polymerase IIA and extensively phosphorylated in RNA polymerase IIO. To investigate the role of the C-terminal domain and the functional significance of its phosphorylation, changes in the level of phosphorylation were followed as a function of the position of RNA polymerase II in the transcription cycle. Complexes were formed with 32P-labeled RNA polymerase IIA and separated from the free polymerase by gel filtration. The phosphorylation state of the RNA polymerase II largest subunit was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Results indicate that RNA polymerase IIA interacts with the template-committed complex to form a stable preinitiation complex. RNA polymerase IIA associated with such complexes is converted to RNA polymerase IIO in the presence of ATP prior to the formation of the first phosphodiester bond. Furthermore, the observation that purified preinitiation complexes can catalyze the conversion of RNA polymerase IIA to IIO indicates that the protein kinase(s) responsible for phosphorylation of the C-terminal domain is a component of such complexes. The concentration of ATP required for the phosphorylation of RNA polymerase II associated with the preinitiation complex is two to three orders of magnitude lower than that required for the conversion of RNA polymerase IIA to IIO free in solution. These results support the idea that phosphorylation of the C-terminal domain of RNA polymerase subunit IIa occurs subsequent to the association of enzyme with the promoter and prior to the initiation of transcription.
Laybourn
P J
PJ
Department of Biochemistry and Biophysics, University of California, Davis 95616.
Dahmus
M E
ME
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D002850:Chromatography, Gel; D006160:Guanosine Triphosphate / Q000378:metabolism; D006367:HeLa Cells / Q000378:metabolism; D006801:Humans; D007527:Isoenzymes / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D011494:Protein Kinases / Q000378:metabolism; D012319:RNA Polymerase II / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D013698:Templates, Genetic; D014158:Transcription, Genetic
D007527:Isoenzymes; D046911:Macromolecular Substances; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D011494:Protein Kinases; D012319:RNA Polymerase II