Genetic information encoded in messenger RNA is translated into protein by the ribosome, which is a large nucleoprotein complex comprising two subunits, denoted 30S and 50S in bacteria. Here we report the crystal structure of the 30S subunit from Thermus thermophilus, refined to 3 A resolution. The final atomic model rationalizes over four decades of biochemical data on the ribosome, and provides a wealth of information about RNA and protein structure, protein-RNA interactions and ribosome assembly. It is also a structural basis for analysis of the functions of the 30S subunit, such as decoding, and for understanding the action of antibiotics. The structure will facilitate the interpretation in molecular terms of lower resolution structural data on several functional states of the ribosome from electron microscopy and crystallography.

The crystal structure of the bacterial 70S ribosome refined to 2.8 angstrom resolution reveals atomic details of its interactions with messenger RNA (mRNA) and transfer RNA (tRNA). A metal ion stabilizes a kink in the mRNA that demarcates the boundary between A and P sites, which is potentially important to prevent slippage of mRNA. Metal ions also stabilize the intersubunit interface. The interactions of E-site tRNA with the 50S subunit have both similarities and differences compared to those in the archaeal ribosome. The structure also rationalizes much biochemical and genetic data on translation.

The 30S ribosomal subunit has two primary functions in protein synthesis. It discriminates against aminoacyl transfer RNAs that do not match the codon of messenger RNA, thereby ensuring accuracy in translation of the genetic message in a process called decoding. Also, it works with the 50S subunit to move the tRNAs and associated mRNA by precisely one codon, in a process called translocation. Here we describe the functional implications of the high-resolution 30S crystal structure presented in the accompanying paper, and infer details of the interactions between the 30S subunit and its tRNA and mRNA ligands. We also describe the crystal structure of the 30S subunit complexed with the antibiotics paromomycin, streptomycin and spectinomycin, which interfere with decoding and translocation. This work reveals the structural basis for the action of these antibiotics, and leads to a model for the role of the universally conserved 16S RNA residues A1492 and A1493 in the decoding process.

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, ...

Elongation factor G (EF-G) is a GTPase that plays a crucial role in the translocation of tRNAs and mRNA during translation by the ribosome. We report a crystal structure refined to 3.6 Å resolution of the ribosome trapped with EF-G in the post-translocational state using the antibiotic fusidic acid. Fusidic acid traps EF-G in a conformation intermediate between the GTP and GDP forms. The interaction of EF-G with ribosomal elements implicated in stimulating catalysis, such as the L10-L12 stalk and the L11 region, and of domain IV of EF-G with P-site tRNA and mRNA shed light on various aspects of EF-G function in catalysis and translocation. The stabilization of the mobile stalks of the ribosome also results in a more complete description of its structure.

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.

We have used the recently determined atomic structure of the 30S ribosomal subunit to determine the structures of its complexes with the antibiotics tetracycline, pactamycin, and hygromycin B. The antibiotics bind to discrete sites on the 30S subunit in a manner consistent with much but not all biochemical data. For each of these antibiotics, interactions with the 30S subunit suggest a mechanism for its effects on ribosome function.

The ribosome selects a correct tRNA for each amino acid added to the polypeptide chain, as directed by mRNA. Aminoacyl-tRNA is delivered to the ribosome by Elongation Factor-Tu (EFTu), which hydrolyzes GTP and releases tRNA in response to codon recognition. The signaling pathway that leads to GTP hydrolysis upon codon recognition is critical to accurate decoding. Here we present the crystal structure of the ribosome complexed with EF-Tu and aminoacyltRNA, refined to 3.6 Å resolution. The structure reveals details of the tRNA distortion that allows aminoacyl-tRNA to interact simultaneously with the decoding center of the 30S subunit and EFTu at the factor-binding site. A series of conformational changes in EF-Tu and aminoacyl-tRNA suggest a communication pathway between the decoding center and the GTPase center of EF-Tu.