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.

Stable m7G Cap-Distal 5′UTR Hairpin Structure Mediates Distinct 40S and 60S Binding Dynamics

“…Translation extracts derived from S. cerevisiae strain YAS1874 (MATa MAK10::URA3 PEP4::HIS3 prb1 prc1 ade2 trp1 his3 ura3) (31) were prepared as previously described (32). Triplet-state quencher-conjugated maleimide dyes (14) were used to label high-salt washed 80S ribosomes and the fluorescently labeled 40S and 60S subunits were purified from high-salt sucrose gradients (33).…”

“…The reconstituted extract was prepared as single-use aliquots, frozen in liquid N 2 , and stored at -80°C. 20 µl of translation mix was assembled fresh by combining 14 µl reconstituted extract with 6 µl of master mix (32). The master mix provides the translation mix and final translation reaction with 2.9 mM and 170 mM of Mg 2+ and K + glutamate salts, respectively.…”

“…All mRNAs were synthesized with the MEGAscript T7 Kit (Ambion) according to the manufacturer's protocol. DNA templates for synthesis of LUC mRNAs were prepared as described previously (32). Synthesis of capCAA AUG , capCAA AAC , and hp-capCAA AAC mRNAs was performed with synthetic DNA templates (IDT) and a clamp DNA oligo (IDT) that was complimentary to the 3′ end of each CAA repeat DNA template and contained a T7 RNA polymerase binding sequence (Supplementary Table 2).…”

“…Data were recorded as a kinetic series at a speed of 2 seconds per frame. Image analyses were performed using custom written Matlab codes, as described previously (32,36). A reconstituted cell-free translation system with fluorescently labeled ribosomal subunits.…”

m7G cap-eIF4E interaction stimulates polysome formation by enhancing first-round initiation kinetics

“…We previously developed a single-molecule assay that enables tracking of single ribosome translation kinetics during active cap-dependent translation (16-18). The assay is based on detection of fluorescently-labeled antibody binding to nascent N-terminal-tagged polypeptide during cell-free translation.…”