title: Phosphorylation of the RNA polymerase II carboxyl-terminal domain by CDK9 is directly responsible for human immunodeficiency virus type 1 Tat-activated transcriptional elongation.
abstract: Stimulation of transcriptional elongation by the human immunodeficiency virus type 1 Tat protein is mediated by CDK9, a kinase that phosphorylates the RNA polymerase II carboxyl-terminal domain (CTD). In order to obtain direct evidence that this phosphorylation event can alter RNA polymerase processivity, we prepared transcription elongation complexes that were arrested by the lac repressor. The CTD was then dephosphorylated by treatment with protein phosphatase 1. The dephosphorylated transcription complexes were able to resume the transcription elongation when IPTG (isopropyl-beta-D-thiogalactopyranoside) and nucleotides were added to the reaction. Under these chase conditions, efficient rephosphorylation of the CTD was observed in complexes containing the Tat protein but not in transcription complexes prepared in the absence of Tat protein. Immunoblots and kinase assays with synthetic peptides showed that Tat activated CDK9 directly since the enzyme and its cyclin partner, cyclin T1, were present at equivalent levels in transcription complexes prepared in the presence or absence of Tat. Chase experiments with the dephosphorylated elongation transcription complexes were performed in the presence of the CDK9 kinase inhibitor DRB (5,6-dichloro-1-beta-D-ribofuranosyl-benzimidazole). Under these conditions there was no rephosphorylation of the CTD during elongation, and transcription through either a stem-loop terminator or bent DNA arrest sequence was strongly inhibited. In experiments in which the CTD was phosphorylated prior to elongation, the amount of readthrough of the terminator sequences was proportional to the extent of the CTD modification. The change in processivity is due to CTD phosphorylation alone, since even after the removal of Spt5, the second substrate for CDK9, RNA polymerase elongation is enhanced by Tat-activated CDK9 activity. We conclude that phosphorylation of the RNA polymerase II CTD by CDK9 enhances transcription elongation directly.
authors.affiliation: Medical Research Council Laboratory of Molecular Biology, Cambridge CB2 2QH, United Kingdom.
authors.forename: Young Kyeung
authors.initials: YK
authors.lastname: Kim
authors.forename: Cyril F
authors.initials: CF
authors.lastname: Bourgeois
authors.forename: Catherine
authors.initials: C
authors.lastname: Isel
authors.forename: Mark J
authors.initials: MJ
authors.lastname: Churcher
authors.forename: Jonathan
authors.initials: J
authors.lastname: Karn
mesh_terms: D001483:Base Sequence; D002868:Chromosomal Proteins, Non-Histone; D042863:Cyclin-Dependent Kinase 9; D018844:Cyclin-Dependent Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D004004:Dichlororibofuranosylbenzimidazole / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015696:Gene Products, tat / Q000235:genetics* / Q000378:metabolism; D015497:HIV-1 / Q000235:genetics* / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007544:Isopropyl Thiogalactoside / Q000737:chemistry / Q000378:metabolism; D008967:Molecular Biology / Q000379:methods; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D012319:RNA Polymerase II / Q000235:genetics / Q000378:metabolism*; D020449:Repetitive Sequences, Amino Acid; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D012694:Serine; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic; D035602:Transcriptional Elongation Factors; D054322:tat Gene Products, Human Immunodeficiency Virus
chemical_list: D002868:Chromosomal Proteins, Non-Histone; D004791:Enzyme Inhibitors; D015696:Gene Products, tat; D010446:Peptide Fragments; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D035602:Transcriptional Elongation Factors; D054322:tat Gene Products, Human Immunodeficiency Virus; C069940:SPT5 transcriptional elongation factor; D007544:Isopropyl Thiogalactoside; D012694:Serine; D004004:Dichlororibofuranosylbenzimidazole; C467969:CDK9 protein, human; D042863:Cyclin-Dependent Kinase 9; D018844:Cyclin-Dependent Kinases; D012319:RNA Polymerase II