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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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tRNA dynamics during frameshifting with the dnaX GCA(Ala) to GUA(Val) mutant mRNA(a) The 3 nucleotides upstream of the slippery sequence (GCA(Ala)) is mutated to GUA(Val) (named the C20U mutant) so that E-site tRNA dynamics can be observed during frameshifting since tRNAVal can be labeled with Cy3-maleimide (see Figure 2). This allows us to estimate the time to translocation during the long rotated-state pause at codon Lys7, since translocation of the Cy3-tRNAVal from the P-site to the E-site leads to rapid departure of the tRNAVal and disappearance of the Cy3 signal. We want to make sure that the C20U mutation does not affect frameshifting dynamics. The nonrotated state and rotated state lifetimes, as well as frameshifting percentages, are consistent with what we have observed before for the wild-type sequence. Number of molecules analyzed n = 266. Error bars, s.e.(b) tRNA-tRNA FRET between the Cy3-tRNAVal in the P site and the incoming Cy5-tRNALys at Lys7 in the A site at the slippery sequence. The tRNAs are in a hybrid state upon encountering of hairpin and engagement with the internal Shine-Dalgarno sequence. After translocation, the Cy3-tRNAVal departs from the ribosome, resulting in a disappearance of FRET. After translocation and uncoupling with ribosome reverse-rotation, the now P-site tRNALys is likely in a hybrid, or “distorted” conformation, according to the structure by Namy et al9. Number of molecules analyzed, top n = 227, bottom n = 337.
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Figure 11: tRNA dynamics during frameshifting with the dnaX GCA(Ala) to GUA(Val) mutant mRNA(a) The 3 nucleotides upstream of the slippery sequence (GCA(Ala)) is mutated to GUA(Val) (named the C20U mutant) so that E-site tRNA dynamics can be observed during frameshifting since tRNAVal can be labeled with Cy3-maleimide (see Figure 2). This allows us to estimate the time to translocation during the long rotated-state pause at codon Lys7, since translocation of the Cy3-tRNAVal from the P-site to the E-site leads to rapid departure of the tRNAVal and disappearance of the Cy3 signal. We want to make sure that the C20U mutation does not affect frameshifting dynamics. The nonrotated state and rotated state lifetimes, as well as frameshifting percentages, are consistent with what we have observed before for the wild-type sequence. Number of molecules analyzed n = 266. Error bars, s.e.(b) tRNA-tRNA FRET between the Cy3-tRNAVal in the P site and the incoming Cy5-tRNALys at Lys7 in the A site at the slippery sequence. The tRNAs are in a hybrid state upon encountering of hairpin and engagement with the internal Shine-Dalgarno sequence. After translocation, the Cy3-tRNAVal departs from the ribosome, resulting in a disappearance of FRET. After translocation and uncoupling with ribosome reverse-rotation, the now P-site tRNALys is likely in a hybrid, or “distorted” conformation, according to the structure by Namy et al9. Number of molecules analyzed, top n = 227, bottom n = 337.

Mentions: We next determined what is occurring during the pause that is characteristic of frameshifting. Normally translocation is coupled to ribosome reverse-rotation with deacylated tRNA in the ribosomal exit site (E site) departing rapidly after the ribosome reverse-rotates16. Using Cy3-labeled tRNAVal, we observed E-site tRNA departure directly at the frameshift site on a GCA21 (Ala) to GUA21 (Val) mRNA mutant, without affecting the frameshifting behavior (Extended Data Fig. 7). We measured the departure of Cy3-tRNAVal relative to the Cy5-tRNALys arrival to the AAA24 (Lys7) codon in the A site, which defines the start of the long rotated-state pause, correlated to peptide bond formation and transition to the rotated state: departure of deacylated Cy3-tRNAVal relative to the arrival of Cy5-tRNALys at codon Lys7 estimates when and if translocation occurs during the pause. During translation of the dnaX mRNA, Cy3-tRNAVal departs on average 45 ± 11 s after the arrival of Cy5-tRNALys to the Lys7 codon (within photobleaching time of 196.7 ± 28.1 s). This time decreases with increasing concentration of EF-G, confirming that tRNA departure is linked to translocation (Fig. 2a). However, since the Cy3-tRNAVal residence time is much shorter than the rotated state lifetime (138 s), translocation occurs within the rotated state pause and precedes eventual reverse rotation. Thus translocation at Lys7 during frameshifting is uncoupled to reverse rotation of the ribosomal subunits. Translocation in this case is still inhibited through the interactions with the hairpin and internal-SD sequence, with time to translocation longer than normal translation.


Dynamic pathways of -1 translational frameshifting.

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

tRNA dynamics during frameshifting with the dnaX GCA(Ala) to GUA(Val) mutant mRNA(a) The 3 nucleotides upstream of the slippery sequence (GCA(Ala)) is mutated to GUA(Val) (named the C20U mutant) so that E-site tRNA dynamics can be observed during frameshifting since tRNAVal can be labeled with Cy3-maleimide (see Figure 2). This allows us to estimate the time to translocation during the long rotated-state pause at codon Lys7, since translocation of the Cy3-tRNAVal from the P-site to the E-site leads to rapid departure of the tRNAVal and disappearance of the Cy3 signal. We want to make sure that the C20U mutation does not affect frameshifting dynamics. The nonrotated state and rotated state lifetimes, as well as frameshifting percentages, are consistent with what we have observed before for the wild-type sequence. Number of molecules analyzed n = 266. Error bars, s.e.(b) tRNA-tRNA FRET between the Cy3-tRNAVal in the P site and the incoming Cy5-tRNALys at Lys7 in the A site at the slippery sequence. The tRNAs are in a hybrid state upon encountering of hairpin and engagement with the internal Shine-Dalgarno sequence. After translocation, the Cy3-tRNAVal departs from the ribosome, resulting in a disappearance of FRET. After translocation and uncoupling with ribosome reverse-rotation, the now P-site tRNALys is likely in a hybrid, or “distorted” conformation, according to the structure by Namy et al9. Number of molecules analyzed, top n = 227, bottom n = 337.
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Figure 11: tRNA dynamics during frameshifting with the dnaX GCA(Ala) to GUA(Val) mutant mRNA(a) The 3 nucleotides upstream of the slippery sequence (GCA(Ala)) is mutated to GUA(Val) (named the C20U mutant) so that E-site tRNA dynamics can be observed during frameshifting since tRNAVal can be labeled with Cy3-maleimide (see Figure 2). This allows us to estimate the time to translocation during the long rotated-state pause at codon Lys7, since translocation of the Cy3-tRNAVal from the P-site to the E-site leads to rapid departure of the tRNAVal and disappearance of the Cy3 signal. We want to make sure that the C20U mutation does not affect frameshifting dynamics. The nonrotated state and rotated state lifetimes, as well as frameshifting percentages, are consistent with what we have observed before for the wild-type sequence. Number of molecules analyzed n = 266. Error bars, s.e.(b) tRNA-tRNA FRET between the Cy3-tRNAVal in the P site and the incoming Cy5-tRNALys at Lys7 in the A site at the slippery sequence. The tRNAs are in a hybrid state upon encountering of hairpin and engagement with the internal Shine-Dalgarno sequence. After translocation, the Cy3-tRNAVal departs from the ribosome, resulting in a disappearance of FRET. After translocation and uncoupling with ribosome reverse-rotation, the now P-site tRNALys is likely in a hybrid, or “distorted” conformation, according to the structure by Namy et al9. Number of molecules analyzed, top n = 227, bottom n = 337.
Mentions: We next determined what is occurring during the pause that is characteristic of frameshifting. Normally translocation is coupled to ribosome reverse-rotation with deacylated tRNA in the ribosomal exit site (E site) departing rapidly after the ribosome reverse-rotates16. Using Cy3-labeled tRNAVal, we observed E-site tRNA departure directly at the frameshift site on a GCA21 (Ala) to GUA21 (Val) mRNA mutant, without affecting the frameshifting behavior (Extended Data Fig. 7). We measured the departure of Cy3-tRNAVal relative to the Cy5-tRNALys arrival to the AAA24 (Lys7) codon in the A site, which defines the start of the long rotated-state pause, correlated to peptide bond formation and transition to the rotated state: departure of deacylated Cy3-tRNAVal relative to the arrival of Cy5-tRNALys at codon Lys7 estimates when and if translocation occurs during the pause. During translation of the dnaX mRNA, Cy3-tRNAVal departs on average 45 ± 11 s after the arrival of Cy5-tRNALys to the Lys7 codon (within photobleaching time of 196.7 ± 28.1 s). This time decreases with increasing concentration of EF-G, confirming that tRNA departure is linked to translocation (Fig. 2a). However, since the Cy3-tRNAVal residence time is much shorter than the rotated state lifetime (138 s), translocation occurs within the rotated state pause and precedes eventual reverse rotation. Thus translocation at Lys7 during frameshifting is uncoupled to reverse rotation of the ribosomal subunits. Translocation in this case is still inhibited through the interactions with the hairpin and internal-SD sequence, with time to translocation longer than normal translation.

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