Crystal structures of the 30S ribosomal subunit in complex with messenger RNA and cognate transfer RNA in the A site, both in the presence and absence of the antibiotic paromomycin, have been solved at between 3.1 and 3.3 angstroms resolution. Cognate transfer RNA (tRNA) binding induces global domain movements of the 30S subunit and changes in the conformation of the universally conserved and essential bases A1492, A1493, and G530 of 16S RNA. These bases interact intimately with the minor groove of the first two base pairs between the codon and anticodon, thus sensing Watson-Crick base-pairing geometry and discriminating against near-cognate tRNA. The third, or "wobble," position of the codon is free to accommodate certain noncanonical base pairs. By partially inducing these structural changes, paromomycin facilitates binding of near-cognate tRNAs.During protein synthesis, the ribosome catalyzes the sequential addition of amino acids to a growing polypeptide chain, using mRNA as a template and aminoacylated tRNAs (aatRNAs) as substrates. Correct base pairing between the three bases of the codon on mRNA and those of the anticodon of the cognate aatRNA dictates the sequence of the polypeptide chain. Discrimination against noncognate tRNA, which generally has two or three mismatches in the base pairing, can be accounted for by the difference in the free energy of base pairing to the codon compared with cognate tRNA. For near-cognate tRNA, which usually involves a single mismatch, the free-energy difference in base pairing compared with cognate tRNA would predict an error rate that is one to two orders of magnitude higher than the actual error rate of protein synthesis (1), and it has long been recognized that the ribosome must improve the accuracy of protein synthesis by discriminating against near-cognate tRNAs (2). This discrimination involves the 30S subunit, which binds mRNA and the anticodon stem-loop (ASL) of tRNA.At the beginning of the elongation cycle, which involves the addition of a new amino acid to a growing polypeptide chain, the aatRNA is presented to the ribosome as a ternary complex with elongation factor Tu (EF-Tu) and guanosine triphosphate (GTP). The selection of cognate tRNA is believed to occur in two stages-an initial recognition step and a proofreading step-that are separated by the irreversible hydrolysis of GTP by EF-Tu (3-6). In this scheme, the discrimination energy inherent in codon-anticodon base pairing is exploited twice to achieve the necessary accuracy. Recent experiments suggest that the binding of cognate rather than near-cognate tRNA results in higher rates of both GTP hydrolysis by EF-Tu, and accommodation, a process in which the acceptor arm of the aa-tRNA swings into the peptidyl transferase site after its release from EF-Tu (7,8). In both steps, the higher rate is proposed to be the result of structural changes in the ribosome induced by cognate tRNA. In the context of proofreading mechanisms alone, it is unclear whether additional structural discrimination by the ribosome, ...

A structural and mechanistic explanation for the selection of tRNAs by the ribosome has been elusive. Here, we report crystal structures of the 30S ribosomal subunit with codon and near-cognate tRNA anticodon stem loops bound at the decoding center and compare affinities of equivalent complexes in solution. In ribosomal interactions with near-cognate tRNA, deviation from Watson-Crick geometry results in uncompensated desolvation of hydrogen-bonding partners at the codon-anticodon minor groove. As a result, the transition to a closed form of the 30S induced by cognate tRNA is unfavorable for near-cognate tRNA unless paromomycin induces part of the rearrangement. We conclude that stabilization of a closed 30S conformation is required for tRNA selection, and thereby structurally rationalize much previous data on translational fidelity.

■ Abstract The underlying basis for the accuracy of protein synthesis has been the subject of over four decades of investigation. Recent biochemical and structural data make it possible to understand at least in outline the structural basis for tRNA selection, in which codon recognition by cognate tRNA results in the hydrolysis of GTP by EF-Tu over 75Å away. The ribosome recognizes the geometry of codonanticodon base pairing at the first two positions but monitors the third, or wobble position, less stringently. Part of the additional binding energy of cognate tRNA is used to induce conformational changes in the ribosome that stabilize a transition state for GTP hydrolysis by EF-Tu and subsequently result in accelerated accommodation of tRNA into the peptidyl transferase center. The transition state for GTP hydrolysis is characterized, among other things, by a distorted tRNA. This picture explains a large body of data on the effect of antibiotics and mutations on translational fidelity. However, many fundamental questions remain, such as the mechanism of activation of GTP hydrolysis by EF-Tu, and the relationship between decoding and frameshifting. CONTENTS

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