Limits...
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.

Show MeSH

Related in: MedlinePlus

tRNALys transit and sampling dynamics(a) Example trace of Cy5-tRNALys transit during translation of the dnaX frameshift mRNA, indicating the definition of pulse lifetime and time between pulse.(b) The time between tRNALys pulse and lifetime of each pulse for the first three Lys codons are consistent with what is expected, and decrease expectedly with the increase of EF-G concentration and tRNAtotal(ΔLys) concentration ([Cy5-tRNALys]= 200 nM). The time between pulse for the first Lys pulse corresponds to the decoding of the first codon Lys1 of the mRNA after 50S subunit joining to the immobilized 30S, which is short as expected and does not depend on factor concentration. The second Lys pulse has a longer time between pulse, corresponding to the ribosome translating four codons from Lys1 to Lys5. The third pulse has a slightly shorter time between pulse, corresponding to the ribosome translating from codon Lys5 to Lys7. The lifetimes for the first two pulses are short, since the ribosomes decode and translocate the corresponding codons normally. The lifetime for the third pulse at codon Lys7 is long, corresponding to the ribosome at the long rotated-state pause during frameshifting. Number of molecules analyzed, n = 179 and n = 212. Error bars, s.e.(c) Mean number of additional tRNALys sampling pulses to the long rotated-state pause at codon Lys7, sampling lifetimes, and sampling arrival times, at various concentrations of EF-G and tRNAtot ([Cy5-tRNALys] = 200 nM) for ribosomes translating the dnaX −1 wild-type frameshifting sequence. There is a mean number of ~2.3 sampling tRNALys pulses, which remains constant at the various factor concentrations. There seems to be an interplay (competition) between EF-G and the other tRNAs on tRNALys sampling. Increasing the concentration of other factors increase the arrival time for tRNALys, probably because they are all competing for the ribosomal A site. Number of molecules analyzed, from left to right, n = 179, n = 212, n = 180, n = 162. Error bars, s.e.(d) The time between tRNALys pulse and lifetime of each pulse for the first three Lys codons are the same for the wild-type AAG sequence and the AAG(AAA) mutant sequence. Number of molecules analyzed n = 212 and n = 454. Error bars, s.e.(e) By translating the AAG(AAA) mutant in the presence of Cy5-tRNALys, we see similar dynamics as the dnaX wild-type sequence. Cy5-tRNALys samples the A site at codon Lys8 after uncoupled translocation at the frameshift site. The fraction of ribosomes exhibiting >4 tRNALys pulses are the same for the wild-type sequence and the AAG(AAA) mutant. The mean number of tRNALys sampling pulses, the mean arrival time, and the mean lifetimes of the sampling pulses to the long rotated-state stall are the same for the AAG(AAA) mutant and the dnaX wild-type sequence. Number of molecules analyzed, n = 212, n = 454. Error bars, s.e.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4472451&req=5

Figure 12: tRNALys transit and sampling dynamics(a) Example trace of Cy5-tRNALys transit during translation of the dnaX frameshift mRNA, indicating the definition of pulse lifetime and time between pulse.(b) The time between tRNALys pulse and lifetime of each pulse for the first three Lys codons are consistent with what is expected, and decrease expectedly with the increase of EF-G concentration and tRNAtotal(ΔLys) concentration ([Cy5-tRNALys]= 200 nM). The time between pulse for the first Lys pulse corresponds to the decoding of the first codon Lys1 of the mRNA after 50S subunit joining to the immobilized 30S, which is short as expected and does not depend on factor concentration. The second Lys pulse has a longer time between pulse, corresponding to the ribosome translating four codons from Lys1 to Lys5. The third pulse has a slightly shorter time between pulse, corresponding to the ribosome translating from codon Lys5 to Lys7. The lifetimes for the first two pulses are short, since the ribosomes decode and translocate the corresponding codons normally. The lifetime for the third pulse at codon Lys7 is long, corresponding to the ribosome at the long rotated-state pause during frameshifting. Number of molecules analyzed, n = 179 and n = 212. Error bars, s.e.(c) Mean number of additional tRNALys sampling pulses to the long rotated-state pause at codon Lys7, sampling lifetimes, and sampling arrival times, at various concentrations of EF-G and tRNAtot ([Cy5-tRNALys] = 200 nM) for ribosomes translating the dnaX −1 wild-type frameshifting sequence. There is a mean number of ~2.3 sampling tRNALys pulses, which remains constant at the various factor concentrations. There seems to be an interplay (competition) between EF-G and the other tRNAs on tRNALys sampling. Increasing the concentration of other factors increase the arrival time for tRNALys, probably because they are all competing for the ribosomal A site. Number of molecules analyzed, from left to right, n = 179, n = 212, n = 180, n = 162. Error bars, s.e.(d) The time between tRNALys pulse and lifetime of each pulse for the first three Lys codons are the same for the wild-type AAG sequence and the AAG(AAA) mutant sequence. Number of molecules analyzed n = 212 and n = 454. Error bars, s.e.(e) By translating the AAG(AAA) mutant in the presence of Cy5-tRNALys, we see similar dynamics as the dnaX wild-type sequence. Cy5-tRNALys samples the A site at codon Lys8 after uncoupled translocation at the frameshift site. The fraction of ribosomes exhibiting >4 tRNALys pulses are the same for the wild-type sequence and the AAG(AAA) mutant. The mean number of tRNALys sampling pulses, the mean arrival time, and the mean lifetimes of the sampling pulses to the long rotated-state stall are the same for the AAG(AAA) mutant and the dnaX wild-type sequence. Number of molecules analyzed, n = 212, n = 454. Error bars, s.e.

Mentions: Uncoupling of tRNA-mRNA translocation from reverse-rotation and E-site tRNA departure creates a non-canonical intermediate in translation: the ribosome has a peptidyl-tRNA in the P site, but remains in a rotated intersubunit conformation. What is the nature of this state and how is it linked to frameshifting? Is the A site available for tRNA binding in this state? To answer these questions, we correlated Cy5-tRNALys binding and departure events with the intersubunit conformational FRET signal (Fig. 2b). Although the dnaX mRNA sequence consists of 4 Lys codons, 71% of elongating ribosomes exhibit >4 Cy5-Lys pulses (Fig. 2c) (equal to frameshifting percentage). The first three tRNALys pulses (Lys1, Lys5, and Lys7) show arrival rates and lifetimes consistent with elongation dynamics from intersubunit FRET data (Extended Data Fig. 8a, b); the third Lys7 pulse corresponds to the ribosome decoding AAA24 Lys7 at the slippery site (lifetime of 119.4 s, consistent with the rotated state lifetime at Lys7). The existence of the fourth and subsequent tRNA pulses directly indicate that translocation has occurred during the long rotated state and the A site is now available for aminoacyl-tRNA binding. After uncoupled translocation of the tRNALys to the P site, which would expose the fourth Lys codon (Lys8), tRNALys samples the A-site codon multiple times (on average ~2.3 times)15, resulting in a buildup of Cy5 intensity (from two Cy5-tRNALys bound to the ribosome, Fig. 2b, c) even though the rotated state is not the natural substrate for tRNA binding to the A site. Mutation of the slippery sequence (A21GA24G) greatly suppresses additional sampling by Cy5-tRNALys (only 9.9% of elongating ribosomes exhibit > 4 pulses), indicating that multiple sampling events on Lys8 are characteristic of frameshifting and the long pause. Postsynchronization of the arrival of the 4th sampling tRNALys to the time of uncoupled translocation shows that translocation gates the arrival of the sampling tRNALys, confirming that tRNALys is indeed sampling the A-site codon exposed by translocation (Fig. 2d, Extended Data Fig. 8). Delivery of tRNA-EF-Tu-GDPNP (a non-hydrolyzable analog of GTP) instead of GTP decreases the tRNA pulse lifetimes from 38 ± 2 s to 2.1 ± 0.1 s, demonstrating that GTP hydrolysis by EF-Tu and subsequent accommodation of the tRNA into the ribosomal A-site occur for these long-lived sampling pulses.


Dynamic pathways of -1 translational frameshifting.

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

tRNALys transit and sampling dynamics(a) Example trace of Cy5-tRNALys transit during translation of the dnaX frameshift mRNA, indicating the definition of pulse lifetime and time between pulse.(b) The time between tRNALys pulse and lifetime of each pulse for the first three Lys codons are consistent with what is expected, and decrease expectedly with the increase of EF-G concentration and tRNAtotal(ΔLys) concentration ([Cy5-tRNALys]= 200 nM). The time between pulse for the first Lys pulse corresponds to the decoding of the first codon Lys1 of the mRNA after 50S subunit joining to the immobilized 30S, which is short as expected and does not depend on factor concentration. The second Lys pulse has a longer time between pulse, corresponding to the ribosome translating four codons from Lys1 to Lys5. The third pulse has a slightly shorter time between pulse, corresponding to the ribosome translating from codon Lys5 to Lys7. The lifetimes for the first two pulses are short, since the ribosomes decode and translocate the corresponding codons normally. The lifetime for the third pulse at codon Lys7 is long, corresponding to the ribosome at the long rotated-state pause during frameshifting. Number of molecules analyzed, n = 179 and n = 212. Error bars, s.e.(c) Mean number of additional tRNALys sampling pulses to the long rotated-state pause at codon Lys7, sampling lifetimes, and sampling arrival times, at various concentrations of EF-G and tRNAtot ([Cy5-tRNALys] = 200 nM) for ribosomes translating the dnaX −1 wild-type frameshifting sequence. There is a mean number of ~2.3 sampling tRNALys pulses, which remains constant at the various factor concentrations. There seems to be an interplay (competition) between EF-G and the other tRNAs on tRNALys sampling. Increasing the concentration of other factors increase the arrival time for tRNALys, probably because they are all competing for the ribosomal A site. Number of molecules analyzed, from left to right, n = 179, n = 212, n = 180, n = 162. Error bars, s.e.(d) The time between tRNALys pulse and lifetime of each pulse for the first three Lys codons are the same for the wild-type AAG sequence and the AAG(AAA) mutant sequence. Number of molecules analyzed n = 212 and n = 454. Error bars, s.e.(e) By translating the AAG(AAA) mutant in the presence of Cy5-tRNALys, we see similar dynamics as the dnaX wild-type sequence. Cy5-tRNALys samples the A site at codon Lys8 after uncoupled translocation at the frameshift site. The fraction of ribosomes exhibiting >4 tRNALys pulses are the same for the wild-type sequence and the AAG(AAA) mutant. The mean number of tRNALys sampling pulses, the mean arrival time, and the mean lifetimes of the sampling pulses to the long rotated-state stall are the same for the AAG(AAA) mutant and the dnaX wild-type sequence. Number of molecules analyzed, n = 212, n = 454. Error bars, s.e.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4472451&req=5

Figure 12: tRNALys transit and sampling dynamics(a) Example trace of Cy5-tRNALys transit during translation of the dnaX frameshift mRNA, indicating the definition of pulse lifetime and time between pulse.(b) The time between tRNALys pulse and lifetime of each pulse for the first three Lys codons are consistent with what is expected, and decrease expectedly with the increase of EF-G concentration and tRNAtotal(ΔLys) concentration ([Cy5-tRNALys]= 200 nM). The time between pulse for the first Lys pulse corresponds to the decoding of the first codon Lys1 of the mRNA after 50S subunit joining to the immobilized 30S, which is short as expected and does not depend on factor concentration. The second Lys pulse has a longer time between pulse, corresponding to the ribosome translating four codons from Lys1 to Lys5. The third pulse has a slightly shorter time between pulse, corresponding to the ribosome translating from codon Lys5 to Lys7. The lifetimes for the first two pulses are short, since the ribosomes decode and translocate the corresponding codons normally. The lifetime for the third pulse at codon Lys7 is long, corresponding to the ribosome at the long rotated-state pause during frameshifting. Number of molecules analyzed, n = 179 and n = 212. Error bars, s.e.(c) Mean number of additional tRNALys sampling pulses to the long rotated-state pause at codon Lys7, sampling lifetimes, and sampling arrival times, at various concentrations of EF-G and tRNAtot ([Cy5-tRNALys] = 200 nM) for ribosomes translating the dnaX −1 wild-type frameshifting sequence. There is a mean number of ~2.3 sampling tRNALys pulses, which remains constant at the various factor concentrations. There seems to be an interplay (competition) between EF-G and the other tRNAs on tRNALys sampling. Increasing the concentration of other factors increase the arrival time for tRNALys, probably because they are all competing for the ribosomal A site. Number of molecules analyzed, from left to right, n = 179, n = 212, n = 180, n = 162. Error bars, s.e.(d) The time between tRNALys pulse and lifetime of each pulse for the first three Lys codons are the same for the wild-type AAG sequence and the AAG(AAA) mutant sequence. Number of molecules analyzed n = 212 and n = 454. Error bars, s.e.(e) By translating the AAG(AAA) mutant in the presence of Cy5-tRNALys, we see similar dynamics as the dnaX wild-type sequence. Cy5-tRNALys samples the A site at codon Lys8 after uncoupled translocation at the frameshift site. The fraction of ribosomes exhibiting >4 tRNALys pulses are the same for the wild-type sequence and the AAG(AAA) mutant. The mean number of tRNALys sampling pulses, the mean arrival time, and the mean lifetimes of the sampling pulses to the long rotated-state stall are the same for the AAG(AAA) mutant and the dnaX wild-type sequence. Number of molecules analyzed, n = 212, n = 454. Error bars, s.e.
Mentions: Uncoupling of tRNA-mRNA translocation from reverse-rotation and E-site tRNA departure creates a non-canonical intermediate in translation: the ribosome has a peptidyl-tRNA in the P site, but remains in a rotated intersubunit conformation. What is the nature of this state and how is it linked to frameshifting? Is the A site available for tRNA binding in this state? To answer these questions, we correlated Cy5-tRNALys binding and departure events with the intersubunit conformational FRET signal (Fig. 2b). Although the dnaX mRNA sequence consists of 4 Lys codons, 71% of elongating ribosomes exhibit >4 Cy5-Lys pulses (Fig. 2c) (equal to frameshifting percentage). The first three tRNALys pulses (Lys1, Lys5, and Lys7) show arrival rates and lifetimes consistent with elongation dynamics from intersubunit FRET data (Extended Data Fig. 8a, b); the third Lys7 pulse corresponds to the ribosome decoding AAA24 Lys7 at the slippery site (lifetime of 119.4 s, consistent with the rotated state lifetime at Lys7). The existence of the fourth and subsequent tRNA pulses directly indicate that translocation has occurred during the long rotated state and the A site is now available for aminoacyl-tRNA binding. After uncoupled translocation of the tRNALys to the P site, which would expose the fourth Lys codon (Lys8), tRNALys samples the A-site codon multiple times (on average ~2.3 times)15, resulting in a buildup of Cy5 intensity (from two Cy5-tRNALys bound to the ribosome, Fig. 2b, c) even though the rotated state is not the natural substrate for tRNA binding to the A site. Mutation of the slippery sequence (A21GA24G) greatly suppresses additional sampling by Cy5-tRNALys (only 9.9% of elongating ribosomes exhibit > 4 pulses), indicating that multiple sampling events on Lys8 are characteristic of frameshifting and the long pause. Postsynchronization of the arrival of the 4th sampling tRNALys to the time of uncoupled translocation shows that translocation gates the arrival of the sampling tRNALys, confirming that tRNALys is indeed sampling the A-site codon exposed by translocation (Fig. 2d, Extended Data Fig. 8). Delivery of tRNA-EF-Tu-GDPNP (a non-hydrolyzable analog of GTP) instead of GTP decreases the tRNA pulse lifetimes from 38 ± 2 s to 2.1 ± 0.1 s, demonstrating that GTP hydrolysis by EF-Tu and subsequent accommodation of the tRNA into the ribosomal A-site occur for these long-lived sampling pulses.

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