Distortion of tRNA upon Near-cognate Codon Recognition on the Ribosome

Joerg Mittelstaet,Andrey L Konevega,Marina V Rodnina

doi:10.1074/jbc.m110.210021

Joerg Mittelstaet, Andrey L Konevega + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m110.210021

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Abstract

The accurate decoding of the genetic information by the ribosome relies on the communication between the decoding center of the ribosome, where the tRNA anticodon interacts with the codon, and the GTPase center of EF-Tu, where GTP hydrolysis takes place. In the A/T state of decoding, the tRNA undergoes a large conformational change that results in a more open, distorted tRNA structure. Here we use a real-time transient fluorescence quenching approach to monitor the timing and the extent of the tRNA distortion upon reading cognate or near-cognate codons. The tRNA is distorted upon codon recognition and remains in that conformation until the tRNA is released from EF-Tu, although the extent of distortion gradually changes upon transition from the pre- to the post-hydrolysis steps of decoding. The timing and extent of the rearrangement is similar on cognate and near-cognate codons, suggesting that the tRNA distortion alone does not provide a specific switch for the preferential activation of GTP hydrolysis on the cognate codon. Thus, although the tRNA plays an active role in signal transmission between the decoding and GTPase centers, other regulators of signaling must be involved.

Highlights

Decoding entails a number of elemental steps
Transient Fluorescence Quenching Approach—To assess the extent of tRNA distortion at the D loop, the binding of the ternary complex EF-Tu1⁄7GTP1⁄7Phe-tRNAPhe(Prf) to the ribosome was followed in a stopped-flow apparatus, monitoring proflavin fluorescence in the presence of increasing concentrations of the fluorescence quencher KI, while keeping the ionic strength constant
To determine the Stern-Volmer quenching constant, KSV, which depends on the accessibility of the fluorophore for the quencher and, is a measure for the “openness” of the tRNA, the differential amplitudes of each step were plotted against the concentration of KI, and the plots were evaluated according to the Stern-Volmer relationship (Equation 1 in “Experimental Procedures”)

Summary

To whom correspondence should be addressed

Takes place codon-independently, mainly through contacts of EF-Tu with ribosomal protein L7/12, and is followed by rapid and reversible codon reading (Fig. 1A; reviewed in Refs. 1– 4). The formation of the fully complementary codon-anticodon duplex induces local and global conformational changes at the decoding center of the ribosome, which lock the aa-tRNA in the codon-bound state and activate EF-Tu for rapid GTP hydrolysis [5,6,7,8]. Binding of near-cognate ternary complexes that entail single mismatches between codon and anticodon does not induce these structural rearrangements or rapid GTP hydrolysis, explaining why initial tRNA selection is more accurate than can be accounted for by the energetic differences between fully matched and mismatched codon-anticodon pairs alone. We study the timeresolved distortion of aa-tRNA at different stages of decoding on cognate and near-cognate codons by monitoring the transient fluorescence quenching in real time

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION