The three-dimensional structure of the translating 70S E. coli ribosome is presented in its two main conformations: the pretranslocational and the posttranslocational states. Using electron cryomicroscopy and angular reconstitution, structures at 20 A resolution were obtained, which, when compared with our earlier reconstruction of "empty" ribosomes, showed densities corresponding to tRNA molecules--at the P and E sites for posttranslocational ribosomes and at the A and P sites for pretranslocational ribosomes. The P-site tRNA lies directly above the bridge connecting the two ribosomal subunits, with the A-site tRNA fitted snugly against it at an angle of approximately 50 degrees, toward the L7/L12 side of the ribosome. The E-site tRNA appears to lie between the side lobe of the 30S subunit and the L1 protuberance.

Synthetic mRNA analogues were prepared by T7 transcription, each containing several thio‐uridine residues at selected positions. After binding to the ribosome in the presence of cognate tRNA, the thio‐U residues were activated by UV irradiation and the resulting sites of cross‐linking to 16S RNA analysed. Three distinct cross‐links were consistently observed: (i) from position ‘+6’ of the mRNA (the 3′‐base of the A‐site codon) to base 1052 of 16S RNA; (ii) from position ‘+7’ of the mRNA to base 1395; and (iii) from ‘+11’ to base 532. Individual yields of the cross‐links were strongly dependent on the particular mRNA sequence in each case. The ‘+11/532’ and ‘+6/1052’ cross‐links were always entirely tRNA‐dependent, whereas the ‘+7/1395’ cross‐link was observed at lower intensity in the absence of tRNA. In the presence of a second (A‐site bound) tRNA the +6/1052 cross‐link was markedly reduced. A cross‐link to the 1050 region was again observed when a message carrying a thio‐U at position ‘+9’ was translocated on the ribosome so as to bring the thio‐U to position +6. Taken together, the data are incompatible with some current models both for the three‐dimensional arrangement of 16S RNA and for the orientation of the tRNA‐mRNA complex in the ribosome.

mRNA analogues approximately 40 bases long were prepared by T7 transcription from synthetic DNA templates. Each message contained the sequence ACC‐GCG (coding for threonine and alanine, respectively), together with a single thio‐U residue located at a variable position on the 3′‐side of these coding triplets. The thio‐U residue was either substituted with 4‐azidophenacyl bromide to introduce a photo‐reactive group, or was left unsubstituted for direct UV cross‐linking. After binding to Escherichia coli 70S ribosomes in the presence of tRNA‐Thr or tRNA‐Ala, the thio‐U residue or azidophenyl group was photo‐activated and the products of cross‐linking (which was exclusively to the 30S subunit) were analysed. Immunological analysis of the cross‐linked proteins showed that S5 and S3, together with S1, were the targets of cross‐linking at positions close to the decoding site, with the cross‐linking to S3 and S1 persisting at positions further away. Analysis of the 16S RNA showed cross‐links to the region of bases 1390–1400 in all cases, but in one instance (with the reactive nucleotide 11 bases from the decoding site) simultaneous cross‐linking was observed to the latter region and to position 532; these two RNA regions are far apart in current three‐dimensional models of the 30S subunit.

Peptides of different lengths encoded by suitable mRNA fragments were biosynthesized in situ on Escherichia coli ribosomes. The peptides carried a diazirine derivative bound to their N-terminal methionine residue, which was photoactivated whilst the peptides were still attached to the ribosome. Subsequently, the sites of photo-cross-linking to 23S RNA were analyzed by our standard procedures. The N-termini of peptides of increasing length became progressively cross-linked to nucleotide 750 (peptides of 6, 9 or 13-15 amino acids), to nucleotide 1614 and concomitantly to a second site between nucleotides 1305 and 1350 (a peptide of 25-26 amino acids), and to nucleotide 91 (a peptide of 29-33 amino acids). Previously we had shown that peptides of 1 or 2 amino acids were cross-linked to nucleotides 2062, 2506 and 2585 within the peptidyl transferase ring, whereas tri-and tetrapeptides were additionally cross-linked to nucleotides 2609 and 1781. Taken together, the data demonstrate that the path of the nascent peptide chain moves from the peptidyl transferase ring in domain V of the 23S RNA to domain IV, then to domain II, then to domain III, and finally to domain I. These cross-linking results are correlated with other types of topographical data relating to the 50S subunit.

mRNA analogues containing 4-thiouridine residues at selected sites were used to extend our analysis of photo-induced cross-links between mRNA and 16S RNA to cover the entire downstream range between positions +1 and +16 on the mRNA (position +1 is the 5'-base of the P-site codon). No tRNA-dependent cross-links were observed from positions +1, +2, +3 or +5. Position +4 on the mRNA was cross-linked in a tRNA-dependent manner to 16S RNA at a site between nucleotides ca 1402-1415 (most probably to the modified residue 1402), and this was absolutely specific for the +4 position. Similarly, the previously observed cross-link to nucleotide 1052 was absolutely specific for the +6 position. The previously observed cross-links from +7 to nucleotide 1395 and from +11 to 532 were however seen to a lesser extent with certain types of mRNA sequence from neighbouring positions (+6 to +10, and +10 to +13, respectively); no tRNA-dependent cross-links to other sites on 16S RNA were found from these positions, and no cross-linking was seen from positions +14 to +16. In each case the effect of a second cognate tRNA (at the ribosomal A-site) on the level of cross-linking was studied, and the specificity of each cross-link was confirmed by translocation experiments with elongation factor G, using appropriate mRNA analogues.