The delivery of a specific amino acid to the translating ribosome is fundamental to protein synthesis. The binding of aminoacyl-transfer RNA to the ribosome is catalysed by the elongation factor Tu (EF-Tu). The elongation factor, the aminoacyl-tRNA and GTP form a stable 'ternary' complex that binds to the ribosome. We have used electron cryomicroscopy and angular reconstitution to visualize directly the kirromycin-stalled ternary complex in the A site of the 70S ribosome of Escherichia coli. Electron cryomicroscopy had previously given detailed ribosomal structures at 25 and 23 A resolution, and was used to determine the position of tRNAs on the ribosome. In particular, the structures of pre-translocational (tRNAs in A and P sites) and post-translocational ribosomes (P and E sites occupied) were both visualized at a resolution of approximately 20 A. Our three-dimensional reconstruction at 18 A resolution shows the ternary complex spanning the inter-subunit space with the acceptor domain of the tRNA reaching into the decoding centre. Domain 1 (the G domain) of the EF-Tu is bound both to the L7/L12 stalk and to the 50S body underneath the stalk, whereas domain 2 is oriented towards the S12 region on the 30S subunit.

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.

Three different mRNA analogues (28 to 34 nucleotides 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 5'-side of these coding triplets. A photo-reactive group was introduced by substitution of the thio-U with 4-azidophenacyl bromide. The messages were bound to E. coli 70S ribosomes in the presence of the appropriate tRNA-Thr or tRNA-Ala, and the azidophenyl group was photoactivated. Cross-linking was found to occur exclusively within the 30S subunit, with the 32P-label in the cross-linked mRNA being divided roughly equally between 30S ribosomal proteins and 16S RNA. Immunological analysis of the cross-linked proteins showed that, in the presence of either tRNA species, protein S7 was the primary target, whereas in the absence of tRNA only small amounts of protein S21 were cross-linked. The cross-link site to 16S RNA lay in all cases very close to its extreme 3'-terminus. These data indicate that the outgoing message leaves the cleft of the 30S subunit in a "northerly" direction.

A DNA fragment containg the Escherichia coUl 5S rDNA sequence linked to a 17 promoter was prepared by PCR from an M13 clone carrying the 5S-complementary sequence. The DNA was transcribed with ¶7 polymerase using a mixture of [a-32P]UTP and 4-thio-UTP, yielding a transcript in which 18% of the uridine residues were randomly replaced by thiouridine. This modified 5S RNA could be reconstituted efficiently into 50S ribosomal subunits or 70S functional complexes. The reconstituted particles were irradiated at wavelengths above 300 nm, and the crosslinked ribosomal components were identified. A crosslink in high yield was reproducibly observed between the modified 5S RNA and 23S RNA, involving residue U-89 of the 5S RNA (at the loop end of helix IV) linked to nucleotide 2477 of the 23S RNA in the loop end of helix 89, immediately adjacent to the peptidyltransferase "ring." On the basis of this result, and in combination with earlier immunoelectron microscopic data, we propose a model for the orientation of the 5S RNA in the 50S subunit.Interactions and neighborhoods between 5S rRNA and other components of the Escherichia coli 50S ribosomal subunit have been investigated in a number of laboratories by a variety of techniques. From protein binding studies (e.g., refs. 1 and 2) the 5S RNA has long been known to interact with proteins L5, L18, and L25, and the binding sites of these proteins on the 5S molecule have been studied in detail by footprinting methods (e.g., refs. 3 and 4). The 5' and 3' ends of the 5S RNA (5-7), as well as residues A-39 and U-40 at the loop end of helix III (8), have been localized by immunoelectron microscopy (IEM) on the central protuberance ofthe 50S subunit, in good agreement with the corresponding IEM locations ofproteins L5, L18, and L25, all of which were also found to lie on the central protuberance (9, 10). On the other hand, very little information is available relating to possible interactions between 5S and 23S RNA (11).Our laboratories have made extensive use of mRNA analogues containing 4-thiouridine (thio-U) residues, in order to study contacts between mRNA and 16S RNA by photocrosslinking (12-15), and the success of this approach has prompted us to apply the same methodology in the search for contacts between 5S and 23S RNA. Accordingly, a modified 5S RNA was constructed containing a random distribution of thio-U residues in place of normal uridine, and the modified molecule was reconstituted into 50S subunits or 70S ribosomes as a substrate for photocrosslinking. The principal result of the subsequent analysis, which we report here, was the characterization of a high-yield crosslink from the loop end of helix IV of the 5S RNA to a site close to the peptidyltransferase ring of the 23S RNA.MATERIALS AND METHODS Construction of Modified 5S RNA. Plasmid pKK223-3 (Pharmacia), which carries the E. coli 5S rDNA sequence, was cut with HindIII and Ssp I to yield a 500-bp fragment encompassing the 5S rDNA; this fragment was cloned by standard procedures (16) into phage M13mpl8 (...