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. 2007 Aug 21;104(34):13661-5.
doi: 10.1073/pnas.0705988104. Epub 2007 Aug 15.

The role of fluctuations in tRNA selection by the ribosome

Tae-Hee Lee  1 , Scott C Blanchard, Harold D Kim, Joseph D Puglisi, Steven Chu
Affiliations

The role of fluctuations in tRNA selection by the ribosome

Tae-Hee Lee et al. Proc Natl Acad Sci U S A. .

Abstract

The detailed mechanism of how the ribosome decodes protein sequence information with an abnormally high accuracy, after 40 years of study, remains elusive. A critical element in selecting correct transfer RNA (tRNA) transferring correct amino acid is "induced fit" between the ribosome and tRNA. By using single-molecule methods, the induced fit mechanism is shown to position favorably the correct tRNA after initial codon recognition. We provide evidence that this difference in positioning and thermal fluctuations constitutes the primary mechanism for the initial selection of tRNA. This work demonstrates thermal fluctuations playing a critical role in the substrate selection by an enzyme.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Schematic of single-molecule FRET experimental setup, examples of resulting FRET traces, and analysis results of cognate (Left) and near-cognate (Right) ternary complex delivery to the ribosome. (A) Single-molecule FRET experimental setup to observe the initial selection of aa-tRNA by the ribosome (8). (B) Typical FRET traces of initial tRNA selection process with the nonhydrolyzable GTP analogue, GDPNP. Materials and methods are explained elsewhere (8). Briefly, the green time trace shows the Cy3 fluorescence intensity, and the orange trace is the Cy5 fluorescence intensity. FRET efficiency is defined as ICy5 /(ICy3 + ICy5). In the presence of GDPNP, aa-tRNA selection process is stalled after the initial selection is complete, thus the system cannot proceed past the mid-FRET state. (C) Postsynchronized histograms and FRET efficiency histograms of cognate and near-cognate delivery of GDPNP stalled ternary complexes (see SI Text). The individual FRET trajectories for the cognate (56 particles) and near-cognate (72 particles) ribosome complexes are superimposed after synchronizing to the first observation of FRET ≥0.27. (Upper) The interaction between the cognate ternary complex and the ribosome results in rapid progression from low- to mid-FRET state, and the majority of near-cognate ribosome complexes retreats from low-FRET state to “0” FRET. (Lower) The mean FRET efficiencies for low- and mid-FRET states in the GDPNP stalled cases. At 15 mM [Mg2+], the mid-FRET efficiencies are 0.433 ± 0.005 and 0.394 ± 0.007 for cognate and near-cognate cases, respectively. The low-FRET efficiencies are 0.330 ± 0.002 and 0.317 ± 0.002 for cognate and near-cognate cases, respectively, showing statistically significant difference in the codon-recognition state. At 5 mM [Mg2+], the mid-FRET efficiencies are 0.438 ± 0.027 and 0.442 ± 0.021 for cognate (59 particles) and near-cognate (55 particles), respectively, and the low-FRET efficiencies are 0.348 ± 0.008 and 0.345 ± 0.004 for cognate and near-cognate cases, respectively (data not shown).
Fig. 2.
Fig. 2.
FRET histogram and lifetime analysis of the GTPase-activated state and the codon-recognition states for cognate (Left) and near-cognate (Right) cases. (A) At 15 mM [Mg2+], the mid-FRET (0.34–0.6 FRET) state lifetime histograms are well fit as double exponential decay by the equations: 526 exp(−t/0.094) + 561 exp(−t/0.31) and 641 exp(−t/0.034) + 207 exp(−t/0.17) for cognate and near-cognate cases, respectively, where time is given in seconds. At 5 mM [Mg2+] (histograms not shown), the fits are 463 exp(−t/0.085) + 483 exp(−t/0.23) and 192 exp(−t/0.051) + 2.17 exp(−t/0.46) for cognate and near-cognate cases, respectively. (B) FRET efficiency and lifetime histograms of the codon-recognition state in the presence of 100 μM tetracycline. Mean FRET efficiency and lifetime of codon-recognition state in cognate cases are 0.29 ± 0.006 and 157 ms whereas those in near-cognate cases are 0.25 ± 0.005 and 116 ms.
Fig. 3.
Fig. 3.
A schematic representation of the kinetic model for the initial selection of aa-tRNA. The 30S subunit (lower rectangle) bound to an mRNA (line across the 30S subunit) and the 50S subunit (upper rectangle) are shown divided into small rectangles corresponding to the A, P, and E sites (23). The selection process begins upon the binding of the ternary complex to the ribosome through the interaction between the EF-Tu and the ribosome. We do not observe initial binding with FRET codon-recognition positions with the ternary complex closer to the P site yielding the low-FRET state (state 2). In its attempt to reach the GTPase-activated state (mid-FRET, state 3), the ribosome complex is observed to reversibly sample the pseudo-GTPase-activated state (mid-FRET, state 3′).
Fig. 4.
Fig. 4.
Summary of kinetics data and calculated selection efficiencies. Selection efficiency is defined as the ratio of d[state 3]cognate /dt to d[state 3]near-cognate /dt. Both the cognate and near-cognate ribosome complexes fluctuate rapidly and reversibly to the pseudo-GTPase-activated state (state 3′) before reaching successfully the bona fide GTPase-activated state (state 3). Preferential selection of the cognate ternary complex by the ribosome is achieved by the product of the higher frequency and the higher success rate of the attempts to reach the GTPase-activated state. Rate constants (units are s−1) are calculated by counting FRET transitions between states as described in the text. Missed events attributable to too short lifetimes were added (see SI Text). By assuming that the initial binding of the ternary complex to the ribosome (formation of state 1 in Fig. 3) is not rate-determining and making steady state approximations about state 2 and state 3′ in Fig. 3, the initial selection efficiencies are calculated to be 6.7 and 2.5 × 102 at 15 mM [Mg2+] and 5 mM [Mg2+], respectively.
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