Activation and reactivation of the RNA polymerase II trigger loop for intrinsic RNA cleavage and catalysis

Transcription
Pavel ČabartCraig D Kaplan

Abstract

In addition to RNA synthesis, multisubunit RNA polymerases (msRNAPs) support enzymatic reactions such as intrinsic transcript cleavage. msRNAP active sites from different species appear to exhibit differential intrinsic transcript cleavage efficiency and have likely evolved to allow fine-tuning of the transcription process. Here we show that a single amino-acid substitution in the trigger loop (TL) of Saccharomyces RNAP II, Rpb1 H1085Y, engenders a gain of intrinsic cleavage activity where the substituted tyrosine appears to participate in acid-base chemistry at alkaline pH for both intrinsic cleavage and nucleotidyl transfer. We extensively characterize this TL substitution for each of these reactions by examining the responses RNAP II enzymes to catalytic metals, altered pH, and factor inputs. We demonstrate that TFIIF stimulation of the first phosphodiester bond formation by RNAP II requires wild type TL function and that H1085Y substitution within the TL compromises or alters RNAP II responsiveness to both TFIIB and TFIIF. Finally, Mn(2+) stimulation of H1085Y RNAP II reveals possible allosteric effects of TFIIB on the active center and cooperation between TFIIB and TFIIF.

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Citations

Jan 2, 2015·Proceedings of the National Academy of Sciences of the United States of America·Yara X MejiaCarlos Bustamante
Jul 8, 2016·Nature·Jeremy G BirdBryce E Nickels
Jul 20, 2016·Proceedings of the National Academy of Sciences of the United States of America·Daria EsyuninaAndrey Kulbachinskiy
Nov 30, 2016·PLoS Genetics·Chenxi QiuCraig D Kaplan
Jan 26, 2017·Nucleic Acids Research·Indranil MalikCraig D Kaplan
Jun 14, 2017·Proceedings of the National Academy of Sciences of the United States of America·Tatiana V MishaninaRobert Landick
Nov 29, 2017·Proceedings of the National Academy of Sciences of the United States of America·Nivedita DamodarenKenji Murakami
Jul 14, 2017·Microbiology and Molecular Biology Reviews : MMBR·Yeva Mirzakhanyan, Paul D Gershon
Feb 28, 2020·EMBO Reports·Xueyuan LengSebastian Marquardt
Mar 1, 2017·The Journal of Biological Chemistry·Nataliya MiropolskayaAndrey Kulbachinskiy
Aug 6, 2021·Nucleic Acids Research·Bing WangIrina Artsimovitch

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