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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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Hetereogeneous frameshift products(a) Two different protein products are possible after −1 frameshifting, dependent on whether peptide bond formation occurs during sampling in the −1 or 0 frame. For the first scenario, tRNA sampling to the last three nucleotides of the slippery sequence (YYZ) redefines the ribosome in the −1 frame (YYY), after which the tRNA dissociates to leave an empty A-site codon. After the long rotated state is reverse-rotated by EF-G, tRNAYYY decodes that codon normally, creating a frameshift product denoted by XXY-YYY. For the second scenario, peptide bond formation occurs after slippage of tRNAYYZ into the −1 frame; peptide-bond formation occurs slowly, since the P- and A-site tRNAs would likely not be positioned correctly in the rotated ribosomal conformation. EF-G would then normally and rapidly resolve the newly-created A/P hybrid state and the ribosome reverse-rotates. In this case, the frameshift product will be denoted by XXY-YYZ.(b) Histogram of the fraction of ribosomes translating to a particular codon for the dnaX −1 frameshift AAG(UUU) mRNA, with a schematic. Since the frameshifting percentage for the AAG(UUU) sequence is low, we see that most of the ribosomes translate up to 12 codons where the 0 frame stop codon is. Though, there is a significant number of ribosomes that translate to 11 codons (~25%), compared to ~5% for our previous experiments. There are two possible scenarios for tRNAPhe sampling to the long rotated-state pause to codon Phe8. In the first case, the tRNAPhe defines the reading frame and falls off, after which the ribosome resolves itself through the action of EF-G, followed by the normal decoding of Phe codon at codon 8. In this case, we get 12 cycles of low-high-low FRET intensity, and hence 12 codons translated by our signal. In the second case, the tRNAPhe defines the reading frame, followed by slow peptide bond formation. After peptide bond formation, the ribosome returns to the canonical hybrid and rotated state, for which EF-G then catalyzes reverse rotation. In this case, one low-high-low FRET cycle is missed, so we count 11 codons translated by our signal. This explains the heterogeneity in frameshift products observed in many frameshift systems28. Number of molecules analyzed n = 353.(c) Example traces of Cy5-tRNAPhe (red) sampling to the long rotated-state pause at codon Lys7 correlated with Cy3B/BHQ conformational FRET signal (green), showing the two possible scenarios for tRNA sampling. Case 1 (as described in part (a)) leads to correlation of tRNA arrival and ribosome rotation after the long rotated state pause whereas case 2 leads to overlap of a tRNAPhe pulse with the reverse-rotation of the long pause. Both scenarios occur when translating the AAG(UUU) mutant, with ~58% of ribosomes for case 1 and 42% for case 2. For the 2nd case, the time between the last Cy5-tRNAPhe arrival and the ribosome reverse-rotation is 27.2 s, much longer than the 7.7 s during normal decoding and translocation, suggesting a slow peptidyltransfer reaction. Our results provide a possible explanation for why heterogeneous frameshifting products are observed in many frameshifting systems. Number of molecules analyzed n = 55.
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Figure 14: Hetereogeneous frameshift products(a) Two different protein products are possible after −1 frameshifting, dependent on whether peptide bond formation occurs during sampling in the −1 or 0 frame. For the first scenario, tRNA sampling to the last three nucleotides of the slippery sequence (YYZ) redefines the ribosome in the −1 frame (YYY), after which the tRNA dissociates to leave an empty A-site codon. After the long rotated state is reverse-rotated by EF-G, tRNAYYY decodes that codon normally, creating a frameshift product denoted by XXY-YYY. For the second scenario, peptide bond formation occurs after slippage of tRNAYYZ into the −1 frame; peptide-bond formation occurs slowly, since the P- and A-site tRNAs would likely not be positioned correctly in the rotated ribosomal conformation. EF-G would then normally and rapidly resolve the newly-created A/P hybrid state and the ribosome reverse-rotates. In this case, the frameshift product will be denoted by XXY-YYZ.(b) Histogram of the fraction of ribosomes translating to a particular codon for the dnaX −1 frameshift AAG(UUU) mRNA, with a schematic. Since the frameshifting percentage for the AAG(UUU) sequence is low, we see that most of the ribosomes translate up to 12 codons where the 0 frame stop codon is. Though, there is a significant number of ribosomes that translate to 11 codons (~25%), compared to ~5% for our previous experiments. There are two possible scenarios for tRNAPhe sampling to the long rotated-state pause to codon Phe8. In the first case, the tRNAPhe defines the reading frame and falls off, after which the ribosome resolves itself through the action of EF-G, followed by the normal decoding of Phe codon at codon 8. In this case, we get 12 cycles of low-high-low FRET intensity, and hence 12 codons translated by our signal. In the second case, the tRNAPhe defines the reading frame, followed by slow peptide bond formation. After peptide bond formation, the ribosome returns to the canonical hybrid and rotated state, for which EF-G then catalyzes reverse rotation. In this case, one low-high-low FRET cycle is missed, so we count 11 codons translated by our signal. This explains the heterogeneity in frameshift products observed in many frameshift systems28. Number of molecules analyzed n = 353.(c) Example traces of Cy5-tRNAPhe (red) sampling to the long rotated-state pause at codon Lys7 correlated with Cy3B/BHQ conformational FRET signal (green), showing the two possible scenarios for tRNA sampling. Case 1 (as described in part (a)) leads to correlation of tRNA arrival and ribosome rotation after the long rotated state pause whereas case 2 leads to overlap of a tRNAPhe pulse with the reverse-rotation of the long pause. Both scenarios occur when translating the AAG(UUU) mutant, with ~58% of ribosomes for case 1 and 42% for case 2. For the 2nd case, the time between the last Cy5-tRNAPhe arrival and the ribosome reverse-rotation is 27.2 s, much longer than the 7.7 s during normal decoding and translocation, suggesting a slow peptidyltransfer reaction. Our results provide a possible explanation for why heterogeneous frameshifting products are observed in many frameshifting systems. Number of molecules analyzed n = 55.

Mentions: Here, we directly tracked translation in real time to follow the dynamics of frameshifting, developing a mechanistic model that embraces prior biochemical and structural studies9,23. We propose that the stochastic interaction of the ribosome with the hairpin helix in an open or closed state, and/or formation of the SD-antiSD pairing interaction represent the shunt to either pausing in the rotated state (which leads to uncoupled translocation) or normal translation24. The long-lived, rotated ribosomal state contains a peptidyl-tRNALys in the P-site and AAG27 codon in the A-site creating a non-canonical intermediate in translation, which is required for frameshifting and likely involves weakened tRNA-mRNA-ribosome contacts25,26 and may involve hyper-rotation as recently proposed27. This state frustrates the normal action of both tRNA-EF-Tu -GTP and EF-G-GTP, accounting for the long pause. The repeated sampling of tRNAs to this state during the long pause allows the binding energy of the codon-anticodon pairing to be used to allow slippage while both P- and A-site tRNAs are on the ribosome and redefine the translational frame. Peptidyl-transfer is slow, since the rotated ribosome likely does not position the two tRNAs correctly for peptidyl transfer to occur efficiently. EF-G eventually resolves the state and continues translation. The competition between slow peptide-bond formation and slow translocation explains heterogeneous protein products in prior frameshifting studies28 (Extended Data Fig. 10). Thus, frameshifting involves both EF-G and tRNA, and occurs at an unconventional point during elongation after translocation but before peptidyltransfer11,29 (Fig. 4). The interplay of mRNA sequence and structure with ribosomal dynamics leads to branchpoints during elongation, creating non-canonical paused states that allow unusual events in elongation. Such states may be a central feature of translational control.


Dynamic pathways of -1 translational frameshifting.

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

Hetereogeneous frameshift products(a) Two different protein products are possible after −1 frameshifting, dependent on whether peptide bond formation occurs during sampling in the −1 or 0 frame. For the first scenario, tRNA sampling to the last three nucleotides of the slippery sequence (YYZ) redefines the ribosome in the −1 frame (YYY), after which the tRNA dissociates to leave an empty A-site codon. After the long rotated state is reverse-rotated by EF-G, tRNAYYY decodes that codon normally, creating a frameshift product denoted by XXY-YYY. For the second scenario, peptide bond formation occurs after slippage of tRNAYYZ into the −1 frame; peptide-bond formation occurs slowly, since the P- and A-site tRNAs would likely not be positioned correctly in the rotated ribosomal conformation. EF-G would then normally and rapidly resolve the newly-created A/P hybrid state and the ribosome reverse-rotates. In this case, the frameshift product will be denoted by XXY-YYZ.(b) Histogram of the fraction of ribosomes translating to a particular codon for the dnaX −1 frameshift AAG(UUU) mRNA, with a schematic. Since the frameshifting percentage for the AAG(UUU) sequence is low, we see that most of the ribosomes translate up to 12 codons where the 0 frame stop codon is. Though, there is a significant number of ribosomes that translate to 11 codons (~25%), compared to ~5% for our previous experiments. There are two possible scenarios for tRNAPhe sampling to the long rotated-state pause to codon Phe8. In the first case, the tRNAPhe defines the reading frame and falls off, after which the ribosome resolves itself through the action of EF-G, followed by the normal decoding of Phe codon at codon 8. In this case, we get 12 cycles of low-high-low FRET intensity, and hence 12 codons translated by our signal. In the second case, the tRNAPhe defines the reading frame, followed by slow peptide bond formation. After peptide bond formation, the ribosome returns to the canonical hybrid and rotated state, for which EF-G then catalyzes reverse rotation. In this case, one low-high-low FRET cycle is missed, so we count 11 codons translated by our signal. This explains the heterogeneity in frameshift products observed in many frameshift systems28. Number of molecules analyzed n = 353.(c) Example traces of Cy5-tRNAPhe (red) sampling to the long rotated-state pause at codon Lys7 correlated with Cy3B/BHQ conformational FRET signal (green), showing the two possible scenarios for tRNA sampling. Case 1 (as described in part (a)) leads to correlation of tRNA arrival and ribosome rotation after the long rotated state pause whereas case 2 leads to overlap of a tRNAPhe pulse with the reverse-rotation of the long pause. Both scenarios occur when translating the AAG(UUU) mutant, with ~58% of ribosomes for case 1 and 42% for case 2. For the 2nd case, the time between the last Cy5-tRNAPhe arrival and the ribosome reverse-rotation is 27.2 s, much longer than the 7.7 s during normal decoding and translocation, suggesting a slow peptidyltransfer reaction. Our results provide a possible explanation for why heterogeneous frameshifting products are observed in many frameshifting systems. Number of molecules analyzed n = 55.
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Figure 14: Hetereogeneous frameshift products(a) Two different protein products are possible after −1 frameshifting, dependent on whether peptide bond formation occurs during sampling in the −1 or 0 frame. For the first scenario, tRNA sampling to the last three nucleotides of the slippery sequence (YYZ) redefines the ribosome in the −1 frame (YYY), after which the tRNA dissociates to leave an empty A-site codon. After the long rotated state is reverse-rotated by EF-G, tRNAYYY decodes that codon normally, creating a frameshift product denoted by XXY-YYY. For the second scenario, peptide bond formation occurs after slippage of tRNAYYZ into the −1 frame; peptide-bond formation occurs slowly, since the P- and A-site tRNAs would likely not be positioned correctly in the rotated ribosomal conformation. EF-G would then normally and rapidly resolve the newly-created A/P hybrid state and the ribosome reverse-rotates. In this case, the frameshift product will be denoted by XXY-YYZ.(b) Histogram of the fraction of ribosomes translating to a particular codon for the dnaX −1 frameshift AAG(UUU) mRNA, with a schematic. Since the frameshifting percentage for the AAG(UUU) sequence is low, we see that most of the ribosomes translate up to 12 codons where the 0 frame stop codon is. Though, there is a significant number of ribosomes that translate to 11 codons (~25%), compared to ~5% for our previous experiments. There are two possible scenarios for tRNAPhe sampling to the long rotated-state pause to codon Phe8. In the first case, the tRNAPhe defines the reading frame and falls off, after which the ribosome resolves itself through the action of EF-G, followed by the normal decoding of Phe codon at codon 8. In this case, we get 12 cycles of low-high-low FRET intensity, and hence 12 codons translated by our signal. In the second case, the tRNAPhe defines the reading frame, followed by slow peptide bond formation. After peptide bond formation, the ribosome returns to the canonical hybrid and rotated state, for which EF-G then catalyzes reverse rotation. In this case, one low-high-low FRET cycle is missed, so we count 11 codons translated by our signal. This explains the heterogeneity in frameshift products observed in many frameshift systems28. Number of molecules analyzed n = 353.(c) Example traces of Cy5-tRNAPhe (red) sampling to the long rotated-state pause at codon Lys7 correlated with Cy3B/BHQ conformational FRET signal (green), showing the two possible scenarios for tRNA sampling. Case 1 (as described in part (a)) leads to correlation of tRNA arrival and ribosome rotation after the long rotated state pause whereas case 2 leads to overlap of a tRNAPhe pulse with the reverse-rotation of the long pause. Both scenarios occur when translating the AAG(UUU) mutant, with ~58% of ribosomes for case 1 and 42% for case 2. For the 2nd case, the time between the last Cy5-tRNAPhe arrival and the ribosome reverse-rotation is 27.2 s, much longer than the 7.7 s during normal decoding and translocation, suggesting a slow peptidyltransfer reaction. Our results provide a possible explanation for why heterogeneous frameshifting products are observed in many frameshifting systems. Number of molecules analyzed n = 55.
Mentions: Here, we directly tracked translation in real time to follow the dynamics of frameshifting, developing a mechanistic model that embraces prior biochemical and structural studies9,23. We propose that the stochastic interaction of the ribosome with the hairpin helix in an open or closed state, and/or formation of the SD-antiSD pairing interaction represent the shunt to either pausing in the rotated state (which leads to uncoupled translocation) or normal translation24. The long-lived, rotated ribosomal state contains a peptidyl-tRNALys in the P-site and AAG27 codon in the A-site creating a non-canonical intermediate in translation, which is required for frameshifting and likely involves weakened tRNA-mRNA-ribosome contacts25,26 and may involve hyper-rotation as recently proposed27. This state frustrates the normal action of both tRNA-EF-Tu -GTP and EF-G-GTP, accounting for the long pause. The repeated sampling of tRNAs to this state during the long pause allows the binding energy of the codon-anticodon pairing to be used to allow slippage while both P- and A-site tRNAs are on the ribosome and redefine the translational frame. Peptidyl-transfer is slow, since the rotated ribosome likely does not position the two tRNAs correctly for peptidyl transfer to occur efficiently. EF-G eventually resolves the state and continues translation. The competition between slow peptide-bond formation and slow translocation explains heterogeneous protein products in prior frameshifting studies28 (Extended Data Fig. 10). Thus, frameshifting involves both EF-G and tRNA, and occurs at an unconventional point during elongation after translocation but before peptidyltransfer11,29 (Fig. 4). The interplay of mRNA sequence and structure with ribosomal dynamics leads to branchpoints during elongation, creating non-canonical paused states that allow unusual events in elongation. Such states may be a central feature of translational control.

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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Related in: MedlinePlus