title: Ribosome excursions during mRNA translocation mediate broad branching of frameshift pathways.
abstract: Programmed ribosomal frameshifting produces alternative proteins from a single transcript. -1 frameshifting occurs on Escherichia coli's dnaX mRNA containing a slippery sequence AAAAAAG and peripheral mRNA structural barriers. Here, we reveal hidden aspects of the frameshifting process, including its exact location on the mRNA and its timing within the translation cycle. Mass spectrometry of translated products shows that ribosomes enter the -1 frame from not one specific codon but various codons along the slippery sequence and slip by not just -1 but also -4 or +2 nucleotides. Single-ribosome translation trajectories detect distinctive codon-scale fluctuations in ribosome-mRNA displacement across the slippery sequence, representing multiple ribosomal translocation attempts during frameshifting. Flanking mRNA structural barriers mechanically stimulate the ribosome to undergo back-and-forth translocation excursions, broadly exploring reading frames. Both experiments reveal aborted translation around mutant slippery sequences, indicating that subsequent fidelity checks on newly adopted codon position base pairings lead to either resumed translation or early termination.
authors.affiliation: Department of Chemistry, University of California, Berkeley, Berkeley, CA 94720, USA.
authors.forename: Shannon
authors.initials: S
authors.lastname: Yan
authors.affiliation: Institute of Molecular and Cellular Biology, National Taiwan University, Taipei 10617, Taiwan.
authors.forename: Jin-Der
authors.initials: JD
authors.lastname: Wen
authors.affiliation: Department of Chemistry, University of California, Berkeley, Berkeley, CA 94720, USA; Jason L. Choy Laboratory of Single-Molecule Biophysics, University of California, Berkeley, Berkeley, CA 94720, USA; QB3 Institute, University of California, Berkeley, Berkeley, CA 94720, USA; Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA; Department of Physics, University of California, Berkeley, Berkeley, CA 94720, USA; Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, CA 94720, USA; Kavli Energy NanoSciences Institute, Berkeley, CA 94720, USA.
authors.forename: Carlos
authors.initials: C
authors.lastname: Bustamante
authors.affiliation: Department of Chemistry, University of California, Berkeley, Berkeley, CA 94720, USA. Electronic address: intinoco@lbl.gov.
authors.forename: Ignacio
authors.initials: I
authors.lastname: Tinoco
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics; D001483:Base Sequence; D004258:DNA Polymerase III / Q000235:genetics; D004926:Escherichia coli / Q000378:metabolism; D016368:Frameshift Mutation; D066298:In Vitro Techniques; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics*; D012270:Ribosomes / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; C103076:DnaX protein, Bacteria; D012333:RNA, Messenger; D004258:DNA Polymerase III