Elongation factor SII contacts the 3'-end of RNA in the RNA polymerase II elongation complex.
Elongation factor SII (also known as TFIIS) is an RNA polymerase II binding protein that allows bypass of template arrest sites by activating a nascent RNA cleavage reaction. Here we show that SII contacts the 3'-end of nascent RNA within an RNA polymerase II elongation complex as detected by photoaffinity labeling. Photocross-linking was dependent upon the presence of SII, incorporation of 4-thio-UMP into RNA, and irradiation and was sensitive to treatment by RNase and proteinase. A transcriptionally active mutant of SII lacking the first 130 amino acids was also cross-linked to the nascent RNA, but SII from Saccharomyces cerevisiae, which is inactive in concert with mammalian RNA polymerase II, failed to become photoaffinity labeled. SII-RNA contact was not detected after a labeled oligoribonucleotide was released from the complex by nascent RNA cleavage, demonstrating that this interaction takes place between elongation complex-associated but not free RNA. This shows that the 3'-end of RNA is near the SII binding site on RNA polymerase II and suggests that SII may activate the intrinsic RNA hydrolysis activity by positioning the transcript in the enzyme's active site.
Powell
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W
Graduate Program in Biochemistry and Molecular Biology, Emory University School of Medicine, Atlanta, Georgia 30322, USA.
Bartholomew
B
B
Reines
D
D
D000345:Affinity Labels / Q000378:metabolism; D000818:Animals; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008274:Magnesium / Q000378:metabolism; D009690:Nucleic Acid Conformation; D012313:RNA / Q000378:metabolism*; D012319:RNA Polymerase II / Q000378:metabolism*; D051381:Rats; D014157:Transcription Factors / Q000378:metabolism*; D035165:Transcription Factors, General; D035602:Transcriptional Elongation Factors; D014542:Uridine Monophosphate / Q000031:analogs & derivatives / Q000378:metabolism; D014544:Uridine Triphosphate / Q000378:metabolism
C101666:4-thiouridine monophosphate; D000345:Affinity Labels; D014157:Transcription Factors; D035165:Transcription Factors, General; D035602:Transcriptional Elongation Factors; C018648:transcription factor S-II; D012313:RNA; D014542:Uridine Monophosphate; D012319:RNA Polymerase II; D008274:Magnesium; D014544:Uridine Triphosphate