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Coordination of -1 programmed ribosomal frameshifting by transcript and nascent chain features revealed by deep mutational scanning

Carmody, Patrick J. and Zimmer, Matthew H. and Kuntz, Charles P. and Harrington, Haley R. and Duckworth, Kate E. and Penn, Wesley D. and Mukhopahyay, Suchetana and Miller, Thomas F., III and Schlebach, Jonathan P. (2021) Coordination of -1 programmed ribosomal frameshifting by transcript and nascent chain features revealed by deep mutational scanning. Nucleic Acids Research, 49 (22). pp. 12943-12954. ISSN 0305-1048. PMCID PMC8682741. doi:10.1093/nar/gkab1172. https://resolver.caltech.edu/CaltechAUTHORS:20210312-124533397

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Abstract

Programmed ribosomal frameshifting (PRF) is a translational recoding mechanism that enables the synthesis of multiple polypeptides from a single transcript. During translation of the alphavirus structural polyprotein, the efficiency of −1PRF is coordinated by a ‘slippery’ sequence in the transcript, an adjacent RNA stem–loop, and a conformational transition in the nascent polypeptide chain. To characterize each of these effectors, we measured the effects of 4530 mutations on −1PRF by deep mutational scanning. While most mutations within the slip-site and stem–loop reduce the efficiency of −1PRF, the effects of mutations upstream of the slip-site are far more variable. We identify several regions where modifications of the amino acid sequence of the nascent polypeptide impact the efficiency of −1PRF. Molecular dynamics simulations of polyprotein biogenesis suggest the effects of these mutations primarily arise from their impacts on the mechanical forces that are generated by the translocon-mediated cotranslational folding of the nascent polypeptide chain. Finally, we provide evidence suggesting that the coupling between cotranslational folding and −1PRF depends on the translation kinetics upstream of the slip-site. These findings demonstrate how −1PRF is coordinated by features within both the transcript and nascent chain.


Item Type:Article
Related URLs:
URLURL TypeDescription
https://doi.org/10.1093/nar/gkab1172DOIArticle
http://www.ncbi.nlm.nih.gov/pmc/articles/pmc8682741/PubMed CentralArticle
https://doi.org/10.1101/2021.03.11.435011DOIDiscussion Paper
ORCID:
AuthorORCID
Zimmer, Matthew H.0000-0002-1437-2636
Kuntz, Charles P.0000-0002-9431-4961
Penn, Wesley D.0000-0001-8930-4091
Mukhopahyay, Suchetana0000-0001-9230-4746
Miller, Thomas F., III0000-0002-1882-5380
Schlebach, Jonathan P.0000-0003-0955-7633
Additional Information:© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Received: 07 April 2021. Revision received: 22 October 2021. Accepted: 10 November 2021. Published: 06 December 2021. We thank Christiane Hassel, David Frank Miller, Jun Liu and Douglas Rusch for technical input and assistance. We also acknowledge the support of the Indiana University Flow Cytometry Core Facility and the Indiana University Center for Genomics and Bioinformatics. Simulations were performed using resources of the National Energy Research Scientific Computing Center (NERSC), a U.S. Department of Energy Office of Science User Facility. FUNDING: National Institute of Allergy and Infectious Diseases (NIAID) [R21AI142383 to J.P.S., in part]; National Institute of General Medical Sciences [R01GM125063 to T.F.M., R01GM138845 to J.P.S.]. Funding for open access charge: National Institute of General Medical Sciences [R01GM138845]. The authors wish it to be known that, in their opinion, the first two authors should be regarded as Joint First Authors. DATA AVAILABILITY. Flow cytometry data are available on FlowRepository (https://flowrepository.org/id/FR-FCM-Z3MG). Sequencing data are available in an NCBI BioProject (https://www.ncbi.nlm.nih.gov/bioproject/720960). Computational scripts for DMS are available on GitHub (https://github.com/schebachlab/DMS-analysis-Carmody). Computational scripts for coarse-grained molecular dynamics simulations of translocon-mediated cotranslational folding are available on GitHub (https://github.com/mhzimmer1/SecCGMD). The structural model of the translocation intermediate is available on ModelArchive (https://www.modelarchive.org/doi/10.5452/ma-n2gxk, Access Code: ohZTM7KNWb). Any other data and/ or analyses will be made freely available by the corresponding authors upon request. Conflict of interest statement. None declared.
Funders:
Funding AgencyGrant Number
NIHR21AI142383
NIHR01GM125063
NIHR01GM138845
Issue or Number:22
PubMed Central ID:PMC8682741
DOI:10.1093/nar/gkab1172
Record Number:CaltechAUTHORS:20210312-124533397
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20210312-124533397
Official Citation:Patrick J Carmody, Matthew H Zimmer, Charles P Kuntz, Haley R Harrington, Kate E Duckworth, Wesley D Penn, Suchetana Mukhopadhyay, Thomas F Miller, III, Jonathan P Schlebach, Coordination of -1 programmed ribosomal frameshifting by transcript and nascent chain features revealed by deep mutational scanning, Nucleic Acids Research, Volume 49, Issue 22, 16 December 2021, Pages 12943–12954, https://doi.org/10.1093/nar/gkab1172
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:108413
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:12 Mar 2021 21:42
Last Modified:21 Dec 2021 22:10

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