biostudies-literatureGaba ANCI NIH HHSMemorial Sloan Kettering Cancer CenterNational Institutes of HealthNIGMS NIH HHSe26https://www.ebi.ac.uk/biostudies/studies/S-EPMC7969011biostudies-literatureUnknown49(5)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 stallinNucleic acids researchSmart-ORF: a single-molecule method for accessing ribosome dynamics in both upstream and main open reading frames.PMC7969011R01 GM121847P30 CA008748R01GM121847Fortune TGaba AQu XWang HfalseSmart-ORF: a single-molecule method for accessing ribosome dynamics in both upstream and main open reading frames.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 stallin2021-01-01T00:00:00Z2021 Mar2025-04-26T00:46:13.283Z2025-04-06T09:48:07.244ZS-EPMC79690113333092110.1093/nar/gkaa1185