Dynamic pathways of −1 translational frameshifting

Abstract

Spontaneous changes in the reading frame of translation are rare (frequency of 10−3 – 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 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 acids2,3. 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 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.

Determinants of the Rate of mRNA Translocation in Bacterial Protein Synthesis

“…In this case, there was virtually no inhibition of the translocation rate by the varying SD-anti-SD interaction strength. Interestingly, recently obtained single-molecule data [27] suggest a 2-fold increase in the lifetime (from 5 to 12 s) of the ratcheted ribosomal state by the presence of an internal SD sequence (− 6.1 kcal/mol) separated from the translocation site by 7 nt, similar to the 6-nt separation of the present work (Fig. 4a).…”

“…Furthermore, the only statistically significant cause of codon-specific variation of the protein elongation rate was due to variations in the free energy of interaction between Shine-Dalgarno (SD)-like sequences in open reading frames (ORFs) of translated mRNAs and the anti-SD sequence of 16S rRNA [26]. This is in line with the previous observation that internal SD sequences promote translational frameshifting and more recent singlemolecule experiments showing that elongation is slowed down downstream of SD-like sequences [27].…”

PATACSDB—the database of polyA translational attenuators in coding sequences

“…As such polyA track sequences may support programed translational frameshifts in such mRNA transcripts giving rise to alternative protein products from those genes. This feature of polyA track genes resembles programmed frameshifting observed in viral genes with slippery sequences however without a need for additional mRNA structures that induces ribosome stalling in known viral transcripts (Chen et al, 2014;Yan et al, 2015). The ultimate control over the production and stability of alternative transcripts from polyA track genes in Eukaryotes would be based on mRNA surveillance mechanisms, mainly non-sense mediated mRNA decay (NMD) or if the kinetic stall persists by no-go mRNA decay (NGD).…”

Amino acid sequence repertoire of the bacterial proteome and the occurrence of untranslatable sequences

“…In all experiments, a mixture of total, aminoacylated E. coli tRNAs was used to provide full coverage for all possible codon uses. We have used this approach previously to characterize stalling of translation induced by SecM and ErmCL peptides, as well as frame-shifting signals (14)(15)(16).…”