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. 2008 Apr 3;452(7187):598-603.
doi: 10.1038/nature06716. Epub 2008 Mar 9.

Following translation by single ribosomes one codon at a time

Jin-Der Wen  1 , Laura Lancaster, Courtney Hodges, Ana-Carolina Zeri, Shige H Yoshimura, Harry F Noller, Carlos Bustamante, Ignacio Tinoco
Affiliations

Following translation by single ribosomes one codon at a time

Jin-Der Wen et al. Nature. .

Abstract

We have followed individual ribosomes as they translate single messenger RNA hairpins tethered by the ends to optical tweezers. Here we reveal that translation occurs through successive translocation--and-pause cycles. The distribution of pause lengths, with a median of 2.8 s, indicates that at least two rate-determining processes control each pause. Each translocation step measures three bases--one codon-and occurs in less than 0.1 s. Analysis of the times required for translocation reveals, surprisingly, that there are three substeps in each step. Pause lengths, and thus the overall rate of translation, depend on the secondary structure of the mRNA; the applied force destabilizes secondary structure and decreases pause durations, but does not affect translocation times. Translocation and RNA unwinding are strictly coupled ribosomal functions.

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Figures

Figure 1
Figure 1. Experimental design
A, The ribosome was stalled at the 5′ side of the mRNA hairpin construct, which was then held between two polystyrene beads coated with anti-digoxigenin antibody (top; approximately 3 μm in diameter) and streptavidin (bottom; approximately 2 μm). The micropipette was substituted by a second laser trap when VE274hp was used for translation. Drawings are schematic and not to scale. B, All the mRNA constructs used in this report share the same sequence and design except for the hairpin (upper case) and flanking regions (lower case). The Shine-Dalgarno sequence (AGGAG) and AUG start codon are highlighted in cyan, the tetraloop of the hairpin in green, Val codon patches in grey and Glu in yellow. The codons chosen to stall the ribosome are boxed. a, S3hp, 60 bp. b, VE60hp, 60 bp. c, VE274hp, 274 bp. The complementary sequence for VE274hp is not shown.
Figure 2
Figure 2. Codon-by-codon translation of VE60hp
a, Extension and force trajectories during translation. The data were collected at 200 Hz (blue traces) and smoothed to 10 Hz (red). Discrete steps are indicated by arrowheads. The 18 nm rip at 163 s corresponds to spontaneous opening of the remaining approximately 18-bp hairpin ahead of the translating ribosome. b, Pairwise distance analysis of the extension trajectory in a from 147 to 157 s after correction for drift. c, The pattern of the translation process illustrated by a close-up view of the 148-150 s region. d, Distribution of translocation times. The translocation times (n = 121) were binned in 0.025 s intervals, and the probability density plotted and fitted to three possible Poisson equations (see Supplementary Information): one step (black line), two steps (blue) and three steps (red), with R2 values of 0.45, 0.87 and 0.95, respectively. The k value from the best fit (red line) is 40 ± 4 s-1. Attempts to fit the distribution to two or three steps with different rate constants gave equal rate constants as the best fit.
Figure 3
Figure 3. Translational trajectories and arrests for VE274hp
Translations were followed on dual-trap optical tweezers. a, Examples of translation trajectories from four different ribosomes. The five horizontal yellow stripes correspond to the Glu patches (see c). b, Close-up views of trajectories. Data were collected at 2 kHz and smoothed to 10 Hz. c, Translation arrests. The positions (relative to the A-site on the ribosome) where translation arrests occur are indicated by short horizontal bars, blue for the wild-type VE274hp and red for the mutant hairpin VE274hp-G2A. The VE274hp-G2A differs from the wild type only in the first internal Shine-Dalgarno-like sequence (boxed by solid green line).
Figure 4
Figure 4. Dwell times and force effect
a, Distribution of dwell times from VE274hp. Dwell-time data (n = 180) are pooled from translations of the first Val patch (V1) for 12 ribosomes with mean dwell times less than 2 s. The distribution (0.5 s bins) is well fitted to a mechanism in which two consecutive reactions take place (red line), with k1 = 0.7 ± 0.2 s-1, k2 = 3.4 ± 1.8 s-1 and R2 = 0.93. b, Force effect on dwell times. The median dwell times in each Val patch are plotted as a function of force (mean force in each Val patch, V1-V4). Data points from the same ribosomes are connected by lines. c, Force effect on long pauses. In this example, the translation paused when the force dropped to about 10 pN. After approximately 1 min of pausing, the force was rapidly raised to 18 pN, and the translation resumed immediately.
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References

    1. Smith SB, Cui Y, Bustamante C. Optical-trap force transducer that operates by direct measurement of light momentum. Methods Enzymol. 2003;361:134–162. - PubMed
    1. Wen J-D, et al. Force unfolding kinetics of RNA using optical tweezers. I. Effects of experimental variables on measured results. Biophys. J. 2007;92:2996–3009. - PMC - PubMed
    1. Moazed D, Noller HF. Interaction of tRNA with 23S rRNA in the ribosomal A, P, and E sites. Cell. 1989;57:585–597. - PubMed
    1. Lancaster L, Noller HF. Involvement of 16S rRNA nucleotides G1338 and A1339 in discrimination of initiator tRNA. Mol. Cell. 2005;20:623–632. - PubMed
    1. Wilson KS, Noller HF. Mapping the position of translational elongation factor EF-G in the ribosome by directed hydroxyl radical probing. Cell. 1998;92:131–139. - PubMed

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