title: Dynamic pathways of -1 translational frameshifting.
abstract: Spontaneous changes in the reading frame of translation are rare (frequency of 10(-3) to 10(-4) per codon), but can be induced by specific features in the messenger RNA (mRNA). In the presence of mRNA secondary structures, a heptanucleotide 'slippery sequence' usually defined by the motif X XXY YYZ, and (in some prokaryotic cases) mRNA sequences that base pair with the 3' end of the 16S ribosomal rRNA (internal Shine-Dalgarno sequences), there is an increased probability that a specific programmed change of frame occurs, wherein the ribosome shifts one nucleotide backwards into an overlapping reading frame (-1 frame) and continues by translating a new sequence of amino acids. Despite extensive biochemical and genetic studies, there is no clear mechanistic description for frameshifting. Here we apply single-molecule fluorescence to track the compositional and conformational dynamics of individual ribosomes at each codon during translation of a frameshift-inducing mRNA from the dnaX gene in Escherichia coli. Ribosomes that frameshift into the -1 frame are characterized by a tenfold longer pause in elongation compared to non-frameshifted ribosomes, which translate through unperturbed. During the pause, interactions of the ribosome with the mRNA stimulatory elements uncouple EF-G catalysed translocation from normal ribosomal subunit reverse-rotation, leaving the ribosome in a non-canonical intersubunit rotated state with an exposed codon in the aminoacyl-tRNA site (A site). tRNA(Lys) sampling and accommodation to the empty A site and EF-G action either leads to the slippage of the tRNAs into the -1 frame or maintains the ribosome into the 0 frame. Our results provide a general mechanistic and conformational framework for -1 frameshifting, highlighting multiple kinetic branchpoints during elongation.
authors.affiliation: 1] Department of Applied Physics, Stanford University, Stanford, California 94305-4090, USA [2] Department of Structural Biology, Stanford University School of Medicine, Stanford, California 94305-5126, USA.
authors.forename: Jin
authors.initials: J
authors.lastname: Chen
authors.affiliation: Department of Structural Biology, Stanford University School of Medicine, Stanford, California 94305-5126, USA.
authors.forename: Alexey
authors.initials: A
authors.lastname: Petrov
authors.affiliation: Department of Structural Biology, Stanford University School of Medicine, Stanford, California 94305-5126, USA.
authors.forename: Magnus
authors.initials: M
authors.lastname: Johansson
authors.affiliation: 1] Department of Applied Physics, Stanford University, Stanford, California 94305-4090, USA [2] Department of Structural Biology, Stanford University School of Medicine, Stanford, California 94305-5126, USA.
authors.forename: Albert
authors.initials: A
authors.lastname: Tsai
authors.affiliation: Department of Structural Biology, Stanford University School of Medicine, Stanford, California 94305-5126, USA.
authors.forename: Seán E
authors.initials: SE
authors.lastname: O'Leary
authors.affiliation: Department of Structural Biology, Stanford University School of Medicine, Stanford, California 94305-5126, USA.
authors.forename: Joseph D
authors.initials: JD
authors.lastname: Puglisi
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics; D003062:Codon / Q000235:genetics; D004258:DNA Polymerase III / Q000235:genetics; D004926:Escherichia coli; D018965:Frameshifting, Ribosomal; D007700:Kinetics; D010441:Peptide Chain Elongation, Translational; D020653:Peptide Elongation Factor G / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism; D016364:Reading Frames / Q000235:genetics; D054657:Ribosome Subunits / Q000737:chemistry / Q000378:metabolism; D012270:Ribosomes / Q000737:chemistry / Q000378:metabolism*; D012399:Rotation; D013997:Time Factors
chemical_list: D001426:Bacterial Proteins; D003062:Codon; C103076:DnaX protein, Bacteria; D020653:Peptide Elongation Factor G; D012333:RNA, Messenger; D012346:RNA, Transfer, Amino Acyl; D004258:DNA Polymerase III