Dynamic pathways of-1 translational frameshifting

Article

关键词: SINGLE-MOLECULE FLUORESCENCE;    TRANSFER-RNA SELECTION;    MESSENGER-RNA;    HYBRID STATE;    REAL-TIME;    COMPOSITIONAL DYNAMICS;    P-SITE;    RIBOSOME;    TRANSLOCATION;    CODON;   

DOI  :  10.1038/nature13428

来源: SCIE

【 摘 要 】

Spontaneous changes in the reading frame of translation are rare (frequency of 10(-3) to 10(-4) per codon)(1), 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 39 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(2,3). 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-Gcatalysed 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.

【 授权许可】

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