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Direct measurement of the mechanical work during translocation by the ribosome.

Liu T, Kaplan A, Alexander L, Yan S, Wen JD, Lancaster L, Wickersham CE, Fredrick K, Fredrik K, Noller H, Tinoco I, Bustamante CJ - Elife (2014)

Bottom Line: Here, we address these questions using optical tweezers to follow translation by individual ribosomes along single mRNA molecules, against an applied force.We find that translocation rates depend exponentially on the force, with a characteristic distance close to the one-codon step, ruling out the existence of sub-steps and showing that the ribosome likely functions as a Brownian ratchet.We show that the ribosome generates ∼13 pN of force, barely sufficient to unwind the most stable structures in mRNAs, thus providing a basis for their regulatory role.

View Article: PubMed Central - PubMed

Affiliation: Jason L Choy Laboratory of Single Molecule Biophysics, University of California, Berkeley, Berkeley, United States Department of Physics, University of California, Berkeley, Berkeley, United States.

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Distribution of the translation bursts and pauses durations for all the measured opposing forces.Left panels: distribution of the bursts durations measured at the forces specified in each graph. Right panels: distributions of the pauses durations. With the exception of the pause duration distribution at 2 pN, for which the amount of data is limited (N = 6), all the distributions are well fitted by a single exponential function. The rates calculated from the distributions (k1, k−1) are indicated together with their 95% confidence interval. These rates are in good agreement with the rates calculated from the mean burst and mean pause durations (Figure 4).DOI:http://dx.doi.org/10.7554/eLife.03406.008
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fig4s1: Distribution of the translation bursts and pauses durations for all the measured opposing forces.Left panels: distribution of the bursts durations measured at the forces specified in each graph. Right panels: distributions of the pauses durations. With the exception of the pause duration distribution at 2 pN, for which the amount of data is limited (N = 6), all the distributions are well fitted by a single exponential function. The rates calculated from the distributions (k1, k−1) are indicated together with their 95% confidence interval. These rates are in good agreement with the rates calculated from the mean burst and mean pause durations (Figure 4).DOI:http://dx.doi.org/10.7554/eLife.03406.008

Mentions: Finally, we turn our attention now to the pauses observed during translation. The duration of the translation bursts (and hence the effective pause entry rate, kp) is independent of the force opposing translocation (Figure 4A). Moreover, the full distribution of burst durations is well described, at all forces, by a single exponential function with nearly identical parameters (Figure 4—figure supplement 1). Likewise, Figure 4B shows that the duration of the pauses (and hence the pause exit rate, k-p) is independent of force. Since the pause duration is also well described by single exponential functions with the same parameters at all the tested forces (Figure 4—figure supplement 1), we conclude that entering into the paused state and exiting from it are both governed by single, force-independent steps. Hence, we can cluster all our measurements into one data set and calculate the entry and exit rates: kp = 0.16 s−1 (0.14, 0.18) and k−p = 0.14 s−1 (0.1, 0.18), where the numbers in parenthesis indicate 95% confidence intervals.10.7554/eLife.03406.007Figure 4.Pause entry and exit rates.


Direct measurement of the mechanical work during translocation by the ribosome.

Liu T, Kaplan A, Alexander L, Yan S, Wen JD, Lancaster L, Wickersham CE, Fredrick K, Fredrik K, Noller H, Tinoco I, Bustamante CJ - Elife (2014)

Distribution of the translation bursts and pauses durations for all the measured opposing forces.Left panels: distribution of the bursts durations measured at the forces specified in each graph. Right panels: distributions of the pauses durations. With the exception of the pause duration distribution at 2 pN, for which the amount of data is limited (N = 6), all the distributions are well fitted by a single exponential function. The rates calculated from the distributions (k1, k−1) are indicated together with their 95% confidence interval. These rates are in good agreement with the rates calculated from the mean burst and mean pause durations (Figure 4).DOI:http://dx.doi.org/10.7554/eLife.03406.008
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4126342&req=5

fig4s1: Distribution of the translation bursts and pauses durations for all the measured opposing forces.Left panels: distribution of the bursts durations measured at the forces specified in each graph. Right panels: distributions of the pauses durations. With the exception of the pause duration distribution at 2 pN, for which the amount of data is limited (N = 6), all the distributions are well fitted by a single exponential function. The rates calculated from the distributions (k1, k−1) are indicated together with their 95% confidence interval. These rates are in good agreement with the rates calculated from the mean burst and mean pause durations (Figure 4).DOI:http://dx.doi.org/10.7554/eLife.03406.008
Mentions: Finally, we turn our attention now to the pauses observed during translation. The duration of the translation bursts (and hence the effective pause entry rate, kp) is independent of the force opposing translocation (Figure 4A). Moreover, the full distribution of burst durations is well described, at all forces, by a single exponential function with nearly identical parameters (Figure 4—figure supplement 1). Likewise, Figure 4B shows that the duration of the pauses (and hence the pause exit rate, k-p) is independent of force. Since the pause duration is also well described by single exponential functions with the same parameters at all the tested forces (Figure 4—figure supplement 1), we conclude that entering into the paused state and exiting from it are both governed by single, force-independent steps. Hence, we can cluster all our measurements into one data set and calculate the entry and exit rates: kp = 0.16 s−1 (0.14, 0.18) and k−p = 0.14 s−1 (0.1, 0.18), where the numbers in parenthesis indicate 95% confidence intervals.10.7554/eLife.03406.007Figure 4.Pause entry and exit rates.

Bottom Line: Here, we address these questions using optical tweezers to follow translation by individual ribosomes along single mRNA molecules, against an applied force.We find that translocation rates depend exponentially on the force, with a characteristic distance close to the one-codon step, ruling out the existence of sub-steps and showing that the ribosome likely functions as a Brownian ratchet.We show that the ribosome generates ∼13 pN of force, barely sufficient to unwind the most stable structures in mRNAs, thus providing a basis for their regulatory role.

View Article: PubMed Central - PubMed

Affiliation: Jason L Choy Laboratory of Single Molecule Biophysics, University of California, Berkeley, Berkeley, United States Department of Physics, University of California, Berkeley, Berkeley, United States.

Show MeSH