Nascent RNA Cleavage by Arrested RNA Polymerase II Does Not Require Upstream Translocation of the Elongation Complex on DNA

• Type of Material: Article
• Authors: Weigang Gu, Emory University; Wade Powell, Emory University; John Mote, Emory University; Daniel Reines, Emory University
• Language: English
• Date: 1993-12-05
• Publisher: American Society for Biochemistry and Molecular Biology
• Publication Version: Final Published Version
• Copyright Statement: © 1993 by The American Society for Biochemistry and Molecular Biology, Inc.
• Title of Journal or Parent Work: Journal of Biological Chemistry
• ISSN: 0021-9258
• Volume: 268
• Issue: 34
• Start Page: 25604
• End Page: 25616
• Grant/Funding Information: This work was supported by National Institutes of Health Grant GM-46331 and American Cancer Society Grant JFRA-394.
• Keywords: ESCHERICHIA-COLI; CALF THYMUS; FACTOR-SII; Biochemistry & Molecular Biology; TERMINATION; Science & Technology; RAT-LIVER; Life Sciences & Biomedicine; TERNARY COMPLEXES; AMANITIN; TRANSCRIPTION ELONGATION; INITIATION FACTOR-ALPHA; WHEAT-GERM
• Research Categories: Chemistry, Biochemistry; Biology, Molecular

Abstract

Obstacles incurred by RNA polymerase II during primary transcript synthesis have been identified in vivo and in vitro. Transcription past these impediments requires SII, an RNA polymerase II-binding protein. SII also activates a nuclease in arrested elongation complexes and this nascent RNA shortening precedes transcriptional readthrough. Here we show that in the presence of SII and nucleotides, transcript cleavage is detected during SII- dependent elongation but not during SII-independent transcription. Thus, under typical transcription conditions, SII is necessary but insufficient to activate RNA cleavage. RNA cleavage could serve to move RNA polymerase II away from the transcriptional impediment and/or permit RNA polymerase II multiple attempts at RNA elongation. By mapping the positions of the 3'-ends of RNAs and the elongation complex on DNA, we demonstrate that upstream movement of RNA polymerase II is not required for limited RNA shortening (seven to nine nucleotides) and reactivation of an arrested complex. Arrested complexes become elongation competent after removal of no more than nine nucleotides from the nascent RNA's 3'-end. Further cleavage of nascent RNA, however, does result in 'backward' translocation of the enzyme. We also show that one round of RNA cleavage is insufficient for full readthrough at an arrest site, consistent with a previously suggested mechanism of SII action.