Smart-ORF: A single-molecule method for accessing ribosome dynamics in both upstream and main open reading frames Journal Article

   

Authors:	Gaba, A.; Wang, H.; Fortune, T.; Qu, X.
Article Title:	Smart-ORF: A single-molecule method for accessing ribosome dynamics in both upstream and main open reading frames
Abstract:	Upstream open reading frame (uORF) translation disrupts scanning 43S flux on mRNA and modulates main open reading frame (mORF) translation efficiency. Current tools, however, have limited access to ribosome dynamics in both upstream and main ORFs of an mRNA. Here, we develop a new two-color in vitro fluorescence assay, Smart-ORF, that monitors individual uORF and mORF translation events in real-time with single-molecule resolution. We demonstrate the utility of Smart-ORF by applying it to uORF-encoded arginine attenuator peptide (AAP)-mediated translational regulation. The method enabled quantification of uORF and mORF initiation efficiencies, 80S dwell time, polysome formation, and the correlation between uORF and mORF translation dynamics. Smart-ORF revealed that AAP-mediated 80S stalling in the uORF stimulates the uORF initiation efficiency and promotes clustering of slower uORF-translating ribosomes. This technology provides a new tool that can reveal previously uncharacterized dynamics of uORF-containing mRNA translation. © The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.
Keywords:	translation initiation; metabolism; gene expression regulation; messenger rna; protein synthesis; rna, messenger; protein biosynthesis; cell free system; arginine; ribosomes; open reading frame; ribosome; open reading frames; procedures; peptide chain initiation, translational; cell-free system; single molecule imaging
Journal Title:	Nucleic Acids Research
Volume:	49
Issue:	5
ISSN:	0305-1048
Publisher:	Oxford University Press
Date Published:	2021-03-18
Start Page:	e26
Language:	English
DOI:	10.1093/nar/gkaa1185
PUBMED:	33330921
PROVIDER:	scopus
PMCID:	PMC7969011
DOI/URL:	https://www.scopus.com/inward/record.uri?eid=2-s2.0-85103226985&doi=10.1093%2fnar%2fgkaa1185&partnerID=40&md5=7bbb4b3e0a9df39bdd3f524b9955bbae
Notes:	Article -- Export Date: 3 May 2021 -- Source: Scopus

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