Spontaneous changes in the reading frame of translation are rare (frequency of 10−3 – 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 (SD) 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 the 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 10-fold 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 catalyzed 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). tRNALys sampling and accommodation to the empty A site either lead to the slippage of the tRNAs into the −1 frame or maintain the ribosome into the 0 frame. Our results provide a general mechanistic and conformational framework for −1 frameshifting, highlighting multiple kinetic branchpoints during elongation.
展开▼
