Uniformity of peptide release is maintained by methylation of release factors

• Type of Material: Article
• Authors: William E. Pierson, Washington University in St. Louis; Eric D. Hoffer, Washington University in St. Louis; Hannah E. Keedy, Washington University in St. Louis; Carrie L. Simms, Washington University in St. Louis; Christine M. Dunham, Washington University in St. Louis; Hani S. Zaher, Washington University in St. Louis
• Language: English
• Date: 2016-09-27
• Publisher: Elsevier
• Publication Version: Author Accepted Manuscript (After Peer Review)
• Copyright Statement: © 2016 The Author(s).
• Final Published Version (URL): https://doi.org/10.1016/j.celrep.2016.08.085
• Title of Journal or Parent Work: Cell Reports
• Volume: 17
• Issue: 1
• Start Page: 11
• End Page: 18
• Grant/Funding Agency: NIH; U.S. Department of Energy; National Institutes of Health; NINDS; National Science Foundation; Pew Scholar in the Biomedical Sciences award; Searle Scholars award
• Grant/Funding Information: This work was supported by the National Institutes of Health (NIH R01GM093278 to CMD, R01GM112641 to HSZ), a Searle Scholars award (to HSZ) and a Pew Scholar in the Biomedical Sciences award (to CMD). The Donald Danforth Plant Science Center Proteomics and Mass Spectrometry Facility is supported the National Science Foundation Grant No. DBI-0922879 for acquisition of the LTQ-Velos Pro Orbitrap LC-MS/MS. The Emory Integrated Proteomics Core is supported by the Neuroscience NINDS Core Facilities (P30NS055077), the Emory University School of Medicine and is one of the Emory Integrated Core Facilities. The X-ray crystallography datasets were collected at the NE-CAT beamlines, which are funded by the National Institute of General Medical Sciences from the NIH (P41 GM103403), and at the SERCAT beamlines. The Pilatus 6M detector on 24-ID-C beam line is funded by a NIHORIP HEI grant (S10 RR029205). This research used resources of the Advanced Photon Source, a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DEAC02-06CH11357.
• Keywords: protein synthesis; catalysis; methylation; peptide release
• Subject - Topics: Molecular biology; Proteins

Abstract

Termination of protein synthesis on the ribosome is catalyzed by release factors (RFs), which share a conserved glycine-glycine-glutamine (GGQ) motif. The glutamine residue is methylated in vivo, but a mechanistic understanding of its contribution to hydrolysis is lacking. Here we show that the modification, apart from increasing the overall rate of termination on all dipeptides, substantially increases the rate of peptide release on a subset of amino acids. In the presence of unmethylated RFs, we measure rates of hydrolysis that are exceptionally slow on proline and glycine residues and ~two orders of magnitude faster in the presence of the methylated factors. Structures of 70S ribosomes bound to methylated RF1 and RF2 reveal that the glutamine side-chain methylation packs against 23S rRNA nucleotide 2451 stabilizing the GGQ motif and placing the side-chain amide of the glutamine towards tRNA. These data provide a framework to understanding how release factor modifications impact termination.