The two steps of poly(A)-dependent termination, pausing and release, can be uncoupled by truncation of the RNA polymerase II carboxyl-terminal repeat domain.
The carboxyl-terminal repeat domain (CTD) of RNA polymerase II is thought to help coordinate events during RNA metabolism. The mammalian CTD consists of 52 imperfectly repeated heptads followed by 10 additional residues at the C terminus. The CTD is required for cleavage and polyadenylation in vitro. We studied poly(A)-dependent termination in vivo using CTD truncation mutants. Poly(A)-dependent termination occurs in two steps, pause and release. We found that the CTD is required for release, the first 25 heptads being sufficient. Neither the final 10 amino acids nor the variant heptads of the second half of the CTD were required. No part of the CTD was required for poly(A)-dependent pausing--the poly(A) signal could communicate directly with the body of the polymerase. By removing the CTD, pausing could be observed without being obscured by release. Poly(A)-dependent pausing appeared to operate by slowing down the polymerase, such as by down-regulation of a positive elongation factor. Although the first 25 heptads supported undiminished poly(A)-dependent termination, they did not efficiently support events near the promoter involved in abortive elongation. However, the second half of the CTD, including the final 10 amino acids, was sufficient for these functions.
Park
Noh Jin
NJ
Department of Chemistry and Biochemistry and Molecular Biology Institute, University of California at Los Angeles, Los Angeles, California 90095-1569, USA.
Tsao
David C
DC
Martinson
Harold G
HG
D000818:Animals; D019556:COS Cells; D006367:HeLa Cells; D006801:Humans; D066298:In Vitro Techniques; D011401:Promoter Regions, Genetic; D017434:Protein Structure, Tertiary; D021122:Protein Subunits; D012319:RNA Polymerase II / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020449:Repetitive Sequences, Amino Acid; D017384:Sequence Deletion; D014158:Transcription, Genetic; D014162:Transfection
D021122:Protein Subunits; D012333:RNA, Messenger; D011994:Recombinant Proteins; D012319:RNA Polymerase II