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Dynamic pathways of -1 translational frameshifting.

Chen J, Petrov A, Johansson M, Tsai A, O'Leary SE, Puglisi JD - Nature (2014)

Bottom Line: 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.

View Article: PubMed Central - PubMed

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.

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.

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Slippery sequence mutation (A21GA24G) decreases frameshifting percentage(a) Example trace of a ribosome translating the A21GA24G mutant mRNA in the presence of 80 nM EF-G and 1 µM tRNAtot. There seems to be a slightly longer pause at codon Lys7.(b) Histogram of the fraction of ribosomes translating to a particular codon for the dnaX −1 frameshift A21GA24G mRNA. Most of the ribosomes translate up to 12 codons where the 0 frame stop codon is. The buildup of ribosomes stalled at codon 9 present during frameshifting disappears. By parsing the number of ribosomes that translate beyond codon 9 and up to codon 9, the frameshifting percentage can be calculated (12%).(c) The rotated-state lifetime. The long stall at Lys7 is decreased with the slippery site mutant, suggesting that the extra-long pause is indeed a result of frameshifting. The slight increase in lifetime at Lys7 is due to the effects of the hairpin and internal Shine-Dalgarno sequence. Number of molecules analyzed n = 230. Error bars, s.e.(d) A UUC(Phe) is introduced in the −1 frame downstream of the slippery site of the A21GA24G mutant, similar to above. The A21GA24G mutation is known to decrease frameshifting efficiency down to background levels19.(e) The nonrotated state lifetime and rotated-state lifetime match with our results using codon counting (see above). In the absence of frameshifting, there is still an increase in rotated state lifetime at codon Lys7, due to the increased energy barrier to translocation by the hairpin and internal Shine-Dalgarno sequence, though this increased lifetime is still much less than the Lys7 rotated state lifetime during frameshifting. Number of molecules analyzed n = 538. Error bars, s.e.(f) Using Cy5-tRNAPhe as a score for frameshifting, frameshifting percentage matches with our previous results. The slippery sequence A21GA24G mutant decreases frameshifting percentage down to background levels. Number of molecules analyzed n = 474, n = 538.
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Figure 10: Slippery sequence mutation (A21GA24G) decreases frameshifting percentage(a) Example trace of a ribosome translating the A21GA24G mutant mRNA in the presence of 80 nM EF-G and 1 µM tRNAtot. There seems to be a slightly longer pause at codon Lys7.(b) Histogram of the fraction of ribosomes translating to a particular codon for the dnaX −1 frameshift A21GA24G mRNA. Most of the ribosomes translate up to 12 codons where the 0 frame stop codon is. The buildup of ribosomes stalled at codon 9 present during frameshifting disappears. By parsing the number of ribosomes that translate beyond codon 9 and up to codon 9, the frameshifting percentage can be calculated (12%).(c) The rotated-state lifetime. The long stall at Lys7 is decreased with the slippery site mutant, suggesting that the extra-long pause is indeed a result of frameshifting. The slight increase in lifetime at Lys7 is due to the effects of the hairpin and internal Shine-Dalgarno sequence. Number of molecules analyzed n = 230. Error bars, s.e.(d) A UUC(Phe) is introduced in the −1 frame downstream of the slippery site of the A21GA24G mutant, similar to above. The A21GA24G mutation is known to decrease frameshifting efficiency down to background levels19.(e) The nonrotated state lifetime and rotated-state lifetime match with our results using codon counting (see above). In the absence of frameshifting, there is still an increase in rotated state lifetime at codon Lys7, due to the increased energy barrier to translocation by the hairpin and internal Shine-Dalgarno sequence, though this increased lifetime is still much less than the Lys7 rotated state lifetime during frameshifting. Number of molecules analyzed n = 538. Error bars, s.e.(f) Using Cy5-tRNAPhe as a score for frameshifting, frameshifting percentage matches with our previous results. The slippery sequence A21GA24G mutant decreases frameshifting percentage down to background levels. Number of molecules analyzed n = 474, n = 538.

Mentions: Elongation of the dnaX mRNA is drastically and abruptly perturbed at codon Lys7. Analysis of rates at each codon revealed a 10-fold increase in the rotated state (waiting for EF-G and translocation) lifetime (96 ± 18 s vs. 5~10 s for the other codons) at Lys7, corresponding to tRNAAla(GCA21)-codon pair in the ribosomal peptidyl-tRNA site (P site) and the newly incorporated tRNALys(AAA24) codon pair in the A site, poised for translocation; nonrotated state lifetimes (waiting for TC and peptide bond formation) remain constant at each codon (Fig. 1b, c, d). Furthermore by partitioning frameshifted vs. non-frameshifted ribosomes, an increased rotated-state lifetime at codon Lys7 is observed only for frameshifted ribosomes (138 ± 31 s); non-frameshifted ribosomes translate through the frameshift site seemingly unaffected (13 ± 4 s) (Fig. 1e, confirmed independently in Extended Data Fig. 2d, and repeated with varying factor concentrations in Extended Data Fig. 5). Disruption of the slippery sequence by changing A21 AAA24 AAG27 to G21 AAG24 AAG27 (A21GA24G mutant) caused an expected decrease in frameshifting efficiency to 12% (background level in our experiments is 3~10%), while drastically decreasing the lifetime at codon Lys7 (25 ± 5 s instead of 96 ± 18 s) (Extended Data Fig. 6). Thus, the long lifetime at codon Lys7 is a hallmark of frameshifting and requires the slippery-site sequence. Partitioning between frameshifted and non-frameshifted ribosomes was assumed to occur during the pause induced by frameshift signal. Instead, we demonstrate that the initial branch point occurs prior to the pause, but all frameshifted ribosomes exhibit a pause.


Dynamic pathways of -1 translational frameshifting.

Chen J, Petrov A, Johansson M, Tsai A, O'Leary SE, Puglisi JD - Nature (2014)

Slippery sequence mutation (A21GA24G) decreases frameshifting percentage(a) Example trace of a ribosome translating the A21GA24G mutant mRNA in the presence of 80 nM EF-G and 1 µM tRNAtot. There seems to be a slightly longer pause at codon Lys7.(b) Histogram of the fraction of ribosomes translating to a particular codon for the dnaX −1 frameshift A21GA24G mRNA. Most of the ribosomes translate up to 12 codons where the 0 frame stop codon is. The buildup of ribosomes stalled at codon 9 present during frameshifting disappears. By parsing the number of ribosomes that translate beyond codon 9 and up to codon 9, the frameshifting percentage can be calculated (12%).(c) The rotated-state lifetime. The long stall at Lys7 is decreased with the slippery site mutant, suggesting that the extra-long pause is indeed a result of frameshifting. The slight increase in lifetime at Lys7 is due to the effects of the hairpin and internal Shine-Dalgarno sequence. Number of molecules analyzed n = 230. Error bars, s.e.(d) A UUC(Phe) is introduced in the −1 frame downstream of the slippery site of the A21GA24G mutant, similar to above. The A21GA24G mutation is known to decrease frameshifting efficiency down to background levels19.(e) The nonrotated state lifetime and rotated-state lifetime match with our results using codon counting (see above). In the absence of frameshifting, there is still an increase in rotated state lifetime at codon Lys7, due to the increased energy barrier to translocation by the hairpin and internal Shine-Dalgarno sequence, though this increased lifetime is still much less than the Lys7 rotated state lifetime during frameshifting. Number of molecules analyzed n = 538. Error bars, s.e.(f) Using Cy5-tRNAPhe as a score for frameshifting, frameshifting percentage matches with our previous results. The slippery sequence A21GA24G mutant decreases frameshifting percentage down to background levels. Number of molecules analyzed n = 474, n = 538.
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Figure 10: Slippery sequence mutation (A21GA24G) decreases frameshifting percentage(a) Example trace of a ribosome translating the A21GA24G mutant mRNA in the presence of 80 nM EF-G and 1 µM tRNAtot. There seems to be a slightly longer pause at codon Lys7.(b) Histogram of the fraction of ribosomes translating to a particular codon for the dnaX −1 frameshift A21GA24G mRNA. Most of the ribosomes translate up to 12 codons where the 0 frame stop codon is. The buildup of ribosomes stalled at codon 9 present during frameshifting disappears. By parsing the number of ribosomes that translate beyond codon 9 and up to codon 9, the frameshifting percentage can be calculated (12%).(c) The rotated-state lifetime. The long stall at Lys7 is decreased with the slippery site mutant, suggesting that the extra-long pause is indeed a result of frameshifting. The slight increase in lifetime at Lys7 is due to the effects of the hairpin and internal Shine-Dalgarno sequence. Number of molecules analyzed n = 230. Error bars, s.e.(d) A UUC(Phe) is introduced in the −1 frame downstream of the slippery site of the A21GA24G mutant, similar to above. The A21GA24G mutation is known to decrease frameshifting efficiency down to background levels19.(e) The nonrotated state lifetime and rotated-state lifetime match with our results using codon counting (see above). In the absence of frameshifting, there is still an increase in rotated state lifetime at codon Lys7, due to the increased energy barrier to translocation by the hairpin and internal Shine-Dalgarno sequence, though this increased lifetime is still much less than the Lys7 rotated state lifetime during frameshifting. Number of molecules analyzed n = 538. Error bars, s.e.(f) Using Cy5-tRNAPhe as a score for frameshifting, frameshifting percentage matches with our previous results. The slippery sequence A21GA24G mutant decreases frameshifting percentage down to background levels. Number of molecules analyzed n = 474, n = 538.
Mentions: Elongation of the dnaX mRNA is drastically and abruptly perturbed at codon Lys7. Analysis of rates at each codon revealed a 10-fold increase in the rotated state (waiting for EF-G and translocation) lifetime (96 ± 18 s vs. 5~10 s for the other codons) at Lys7, corresponding to tRNAAla(GCA21)-codon pair in the ribosomal peptidyl-tRNA site (P site) and the newly incorporated tRNALys(AAA24) codon pair in the A site, poised for translocation; nonrotated state lifetimes (waiting for TC and peptide bond formation) remain constant at each codon (Fig. 1b, c, d). Furthermore by partitioning frameshifted vs. non-frameshifted ribosomes, an increased rotated-state lifetime at codon Lys7 is observed only for frameshifted ribosomes (138 ± 31 s); non-frameshifted ribosomes translate through the frameshift site seemingly unaffected (13 ± 4 s) (Fig. 1e, confirmed independently in Extended Data Fig. 2d, and repeated with varying factor concentrations in Extended Data Fig. 5). Disruption of the slippery sequence by changing A21 AAA24 AAG27 to G21 AAG24 AAG27 (A21GA24G mutant) caused an expected decrease in frameshifting efficiency to 12% (background level in our experiments is 3~10%), while drastically decreasing the lifetime at codon Lys7 (25 ± 5 s instead of 96 ± 18 s) (Extended Data Fig. 6). Thus, the long lifetime at codon Lys7 is a hallmark of frameshifting and requires the slippery-site sequence. Partitioning between frameshifted and non-frameshifted ribosomes was assumed to occur during the pause induced by frameshift signal. Instead, we demonstrate that the initial branch point occurs prior to the pause, but all frameshifted ribosomes exhibit a pause.

Bottom Line: 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.

View Article: PubMed Central - PubMed

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.

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.

Show MeSH
Related in: MedlinePlus