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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. . (Unpublished) https://resolver.caltech.edu/CaltechAUTHORS:20210312-124533397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. In the alphavirus structural polyprotein, -1PRF is coordinated by a slippery sequence in the transcript, an RNA stem-loop, and a conformational transition in the nascent polypeptide chain. To characterize each of these effectors, we measured the effects of 4,530 mutations on -1PRF by deep mutational scanning. While most mutations within the slip-site and stem-loop disrupt -1PRF, mutagenic effects upstream of the slip-site are far more variable. Molecular dynamics simulations of polyprotein biogenesis suggest many of these mutations alter stimulatory forces on the nascent chain through their effects on translocon-mediated cotranslational folding. Finally, we provide evidence suggesting 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:Report or Paper (Discussion Paper)
Related URLs:
URLURL TypeDescription
https://doi.org/10.1101/2021.03.11.435011DOIDiscussion Paper
ORCID:
AuthorORCID
Zimmer, Matthew H.0000-0002-1437-2636
Penn, Wesley D.0000-0001-8930-4091
Miller, Thomas F., III0000-0002-1882-5380
Schlebach, Jonathan P.0000-0003-0955-7633
Additional Information:The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license. This version posted March 11, 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. This research was supported in part by grant from the National Institute of Allergy and Infectious Diseases (NIAID) to J. P. S. (R21AI142383) as well as from grants from the National Institute of General Medical Sciences to T. F. M. (R01GM125063) and J. P. S. (R01GM138845). 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. Author Contributions: J.P.S, W.D.P., S.M., and T.F.M. III designed the experiments. P.J.C., H.R.H., K.E.D., and W.D.P produced the genetic constructs and carried out the cellular and genetic experiments. C.P.K. and P.J.C. analyzed and curated the DMS data. M.H.Z. carried out and analyzed CGMD simulations. J.P.S. wrote the manuscript with editorial input from the other authors. The authors declare no conflict of interest.
Funders:
Funding AgencyGrant Number
NIHR21AI142383
NIHR01GM125063
NIHR01GM138845
Record Number:CaltechAUTHORS:20210312-124533397
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20210312-124533397
Official Citation:Coordination of -1 Programmed Ribosomal Frameshifting by Transcript and Nascent Chain Features Revealed by Deep Mutational Scanning. Patrick J. Carmody, Matthew H. Zimmer, Charles P. Kuntz, Haley R. Harrington, Kate E. Duckworth, Wesley D. Penn, Suchetana Mukhopahyay, Thomas F. Miller III, Jonathan P. Schlebach. bioRxiv 2021.03.11.435011; doi: https://doi.org/10.1101/2021.03.11.435011
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:12 Mar 2021 21:42

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