We describe the crystal structure of the complete Thermus thermophilus 70S ribosome containing bound messenger RNA and transfer RNAs (tRNAs) at 5.5 angstrom resolution. All of the 16S, 23S, and 5S ribosomal RNA (rRNA) chains, the A-, P-, and E-site tRNAs, and most of the ribosomal proteins can be fitted to the electron density map. The core of the interface between the 30S small subunit and the 50S large subunit, where the tRNA substrates are bound, is dominated by RNA, with proteins located mainly at the periphery, consistent with ribosomal function being based on rRNA. In each of the three tRNA binding sites, the ribosome contacts all of the major elements of tRNA, providing an explanation for the conservation of tRNA structure. The tRNAs are closely juxtaposed with the intersubunit bridges, in a way that suggests coupling of the 20 to 50 angstrom movements associated with tRNA translocation with intersubunit movement.

The complete nucleotide sequence of the 16S RNA gene from the rrnB cistron of Escherichia coli has been determined by using three rapid DNA sequencing methods. Nearly all of the structure has been confirmed by two to six independent sequence determinations on both DNA strands. The length of the 16S rRNA chain inferred from the DNA sequence is 1541 nucleotides, in close agreement with previous estimates. We note discrepancies between this sequence and the most recent version of it reported from direct RNA sequencing [Ehresmann, C., Stiegler, P., Carbon, P. & Ebel, J. P. (1977) FEBS Lett. 84, 337-341]. A few of these may be explained by heterogeneity among 16S rRNA sequences from different cistrons. No nucleotide sequences were found in the 16S rRNA gene that cannot be reconciled with RNase digestion products of mature 16S rRNA. rRNA is becoming increasingly important in our current perception of the mechanism of action of ribosomes. In particular, we wish to understand more fully the functional role of 16S rRNA, the major molecular component of the small ribosomal subunit of prokaryotes. 16S rRNA has been directly implicated in discrimination of mRNA initiation sites (1, 2), tRNA binding (3, 4), and association of the two ribosomal subunits (5, 6). Full understanding of the workings of the ribosome consequently are becoming limited by our lack of knowledge of rRNA structure. Such information will also be essential for elucidation of the process by which these complex structures assemble themselves. Finally, we expect to gain insight into such diverse problems as protein-nucleic acid recognition and the evolutionary origin of the coding process from a more thorough knowledge of rRNA structure.Partial nucleotide sequences for 16S RNA have been published (7-9). However, numerous discrepancies in oligonucleotide sequences (6, 10, 11) and in the ordering of oligonucleotides (6,(12)(13)(14) have been reported by other investigators. Additional evidence for sequence errors comes from restriction endonuclease mapping of a 16S RNA gene: some cleavage sites predicted from the published sequences were not found (unpublished data). Finally, chemical (6,12,15) and enzymatic (7,8,16) probes have been used to detect single-stranded regions of 16S RNA; the lack of agreement of these findings with the proposed secondary structure derived from the published sequences further suggests error in the primary structure. In view of the possible misinterpretation of various biochemical studies involving 16S RNA, we were prompted to reinvestigate its primary structure.The availability of rapid DNA sequence methods made possible the derivation of the nucleotide sequence of 16S RNA by direct sequencing of the 16S rRNA gene from the rrnB cistron of Escherichia coli. We present here the complete sequence of the portion of the cistron corresponding to mature 16S RNA. Reported discrepancies with the previously published sequence have been confirmed, and additional errors have been found involving oligonucleotide sequences, orderin...

No abstract

Chemical footprinting shows that several classes of antibiotics (streptomycin, tetracycline, spectinomycin, edeine, hygromycin and the neomycins) protect concise sets of highly conserved nucleotides in 16S ribosomal RNA when bound to ribosomes. These findings have strong implications for the mechanism of action of these antibiotics and for the assignment of functions to specific structural features of 16S rRNA.

During the elongation cycle, tRNA and mRNA undergo coupled translocation through the ribosome catalyzed by elongation factor G (EF-G). Cryo-EM reconstructions of certain EF-G-containing complexes led to the proposal that the mechanism of translocation involves rotational movement between the two ribosomal subunits. Here, using single-molecule FRET, we observe that pretranslocation ribosomes undergo spontaneous intersubunit rotational movement in the absence of EF-G, fluctuating between two conformations corresponding to the classical and hybrid states of the translocational cycle. In contrast, posttranslocation ribosomes are fixed predominantly in the classical, nonrotated state. Movement of the acceptor stem of deacylated tRNA into the 50S E site and EF-G binding to the ribosome both contribute to stabilization of the rotated, hybrid state. Furthermore, the acylation state of P site tRNA has a dramatic effect on the frequency of intersubunit rotation. Our results provide direct evidence that the intersubunit rotation that underlies ribosomal translocation is thermally driven.

Direct chemical 'footprinting' shows that translocation of transfer RNA occurs in two discrete steps. During the first step, which occurs spontaneously after the formation of the peptide bond, the acceptor end of tRNA moves relative to the large ribosomal subunit resulting in 'hybrid states' of binding. During the second step, which is promoted by elongation factor EF-G, the anticodon end of tRNA, along with the messenger RNA, moves relative to the small ribosomal subunit.

At termination of protein synthesis, type I release factors promote hydrolysis of the peptidyl-transfer RNA linkage in response to recognition of a stop codon. Here we describe the crystal structure of the Thermus thermophilus 70S ribosome in complex with the release factor RF1, tRNA and a messenger RNA containing a UAA stop codon, at 3.2 A resolution. The stop codon is recognized in a pocket formed by conserved elements of RF1, including its PxT recognition motif, and 16S ribosomal RNA. The codon and the 30S subunit A site undergo an induced fit that results in stabilization of a conformation of RF1 that promotes its interaction with the peptidyl transferase centre. Unexpectedly, the main-chain amide group of Gln 230 in the universally conserved GGQ motif of the factor is positioned to contribute directly to peptidyl-tRNA hydrolysis.

Our understanding of the mechanism of protein synthesis has undergone rapid progress in recent years as a result of low-resolution X-ray and cryo-EM structures of ribosome functional complexes and high-resolution structures of ribosomal subunits and vacant ribosomes. Here, we present the crystal structure of the Thermus thermophilus 70S ribosome containing a model mRNA and two tRNAs at 3.7 A resolution. Many structural details of the interactions between the ribosome, tRNA, and mRNA in the P and E sites and the ways in which tRNA structure is distorted by its interactions with the ribosome are seen. Differences between the conformations of vacant and tRNA-bound 70S ribosomes suggest an induced fit of the ribosome structure in response to tRNA binding, including significant changes in the peptidyl-transferase catalytic site.