TY - JOUR
T1 - Smart-ORF
T2 - A single-molecule method for accessing ribosome dynamics in both upstream and main open reading frames
AU - Gaba, Anthony
AU - Wang, Hongyun
AU - Fortune, Trinisia
AU - Qu, Xiaohui
N1 - Publisher Copyright:
© 2021 The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.
PY - 2021/3/18
Y1 - 2021/3/18
N2 - 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.
AB - 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.
UR - https://www.scopus.com/pages/publications/85103226985
U2 - 10.1093/nar/gkaa1185
DO - 10.1093/nar/gkaa1185
M3 - Article
C2 - 33330921
AN - SCOPUS:85103226985
SN - 0305-1048
VL - 49
JO - Nucleic Acids Research
JF - Nucleic Acids Research
IS - 5
ER -