A nonproteolytic function of the 19S regulatory subunit of the 26S proteasome is required for efficient activated transcription by human RNA polymerase II.
We recently reported that the 19S regulatory subunit of the yeast 26S proteasome stimulates transcription elongation by RNA polymerase II. However, because of basic differences between yeast and mammals in the components and cellular location of the proteasome, it is crucial to assess whether this is a general phenomenon. Here we address this question and demonstrate that (1) the nonproteolytic activity of the 19S (PA700) complex of the proteasome is required for efficient activated transcription in the mammalian in vitro system, (2) this requirement applies to both natural and artificial activators, and (3) highly purified PA700 can provide this activity. In vitro transcription assays using HeLa cell nuclear extracts reveal that antibodies against human Trip1p/Rpt6 (mammalian Sug1p), one of the six ATPases in the PA700, significantly inhibit activated transcription. Similarly, immunodepletion of the PA700 from the extract also significantly reduces activated, but not basal, transcription and add-back of the highly purified mammalian PA700 restores the activity. Finally, inhibitors of the proteasome's peptidase activities do not affect transcription although the peptidase activity is almost completely inhibited. These findings indicate that the requirement for a nonproteolytic activity of the 19S complex in transcription is general in eukaryotes.
Ferdous
Anwarul
A
Center for Biomedical Inventions, Department of Internal Medicine, University of Texas-Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, Texas 75390-8573, USA.
Kodadek
Thomas
T
Johnston
Stephen Albert
SA
D000074183:ATPases Associated with Diverse Cellular Activities; D048868:Adaptor Proteins, Signal Transducing; D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors / Q000276:immunology / Q000378:metabolism / Q000502:physiology*; D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D001666:Binding Sites, Antibody; D002417:Cattle; D010450:Endopeptidases / Q000276:immunology / Q000378:metabolism / Q000502:physiology*; D006367:HeLa Cells; D006801:Humans; D006868:Hydrolysis; D060588:LIM Domain Proteins; D010447:Peptide Hydrolases / Q000276:immunology / Q000378:metabolism / Q000502:physiology*; D046988:Proteasome Endopeptidase Complex; D021122:Protein Subunits; D012319:RNA Polymerase II / Q000037:antagonists & inhibitors / Q000502:physiology*; D014157:Transcription Factors / Q000037:antagonists & inhibitors / Q000276:immunology; D015533:Transcriptional Activation / Q000502:physiology*
D048868:Adaptor Proteins, Signal Transducing; D000911:Antibodies, Monoclonal; D060588:LIM Domain Proteins; C092282:PSMC5 protein, human; D021122:Protein Subunits; D014157:Transcription Factors; D012319:RNA Polymerase II; D010450:Endopeptidases; D010447:Peptide Hydrolases; D046988:Proteasome Endopeptidase Complex; C061553:ATP dependent 26S protease; C119011:26S proteasome non-ATPase regulatory subunit 13; D000251:Adenosine Triphosphatases; D000074183:ATPases Associated with Diverse Cellular Activities