Visualization of elongation factor Tu on the Escherichia coli ribosome.

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.

A new model for the three-dimensional folding of Escherichia coli 16 S ribosomal RNA. III. The topography of the functional centre.

We describe the locations of sites within the 3D model for the 16 S rRNA (described in two accompanying papers) that are implicated in ribosomal function. The relevant experimental data originate from many laboratories and include sites of foot-printing, cross-linking or mutagenesis for various functional ligands. A number of the sites were themselves used as constraints in building the 16 S model. (1) The foot-print sites for A site tRNA are all clustered around the anticodon stem-loop of the tRNA; there is no "allosteric" site. (2) The foot-print sites for P site tRNA that are essential for P site binding are similarly clustered around the P site anticodon stem-loop. The foot-print sites in 16 S rRNA helices 23 and 24 are, however, remote from the P site tRNA. (3) Cross-link sites from specific nucleotides within the anticodon loops of A or P site-bound tRNA are mostly in agreement with the model, whereas those from nucleotides in the elbow region of the tRNA (which also exhibit extensive cross-linking to the 50 S subunit) are more widely spread. Again, cross-links to helix 23 are remote from the tRNAs. (4) The corresponding cross-links from E site tRNA are predominantly in helix 23, and these agree with the model. Electron microscopy data are presented, suggestive of substantial conformational changes in this region of the ribosome. (5) Foot-prints for IF-3 in helices 23 and 24 are at a position with close contact to the 50 S subunit. (6) Foot-prints from IF-1 form a cluster around the anticodon stem-loop of A site tRNA, as do also the sites on 16 S rRNA that have been implicated in termination. (7) Foot-print sites and mutations relating to streptomycin form a compact group on one side of the A site anticodon loop, with the corresponding sites for spectinomycin on the other side. (8) Site-specific cross-links from mRNA (which were instrumental in constructing the 16 S model) fit well both in the upstream and downstream regions of the mRNA, and indicate that the incoming mRNA passes through the well-defined "hole" at the head-body junction of the 30 S subunit.

Arrangement of tRNAs in pre- and posttranslocational ribosomes revealed by electron cryomicroscopy.

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.

The ribosomal environment of tRNA: crosslinks to rRNA from positions 8 and 20:1 in the central fold of tRNA located at the A, P, or E site.

The naturally occurring nucleotide 3-(3-amino-3-carboxy-propyl) uridine ("acp3U") at position 20:1 of lupin tRNAMet was coupled to a photoreactive diazirine derivative. Similarly, the 4-thiouridine at position 8 of Escherichia coli tRNAPhe was modified with an aromatic azide. Each of the derivatized tRNAs was bound to E. coli ribosomes in the presence of suitable mRNA analogues, under conditions specific for the A, P, or E sites. After photoactivation of the diazirine or azide groups, the sites of crosslinking from the tRNAs to 16S or 23S rRNA were analyzed by our standard procedures, involving a combination of ribonuclease H digestion and primer extension analysis. The crosslinked ribosomal proteins were also identified. The results for the rRNA showed a well-defined series of crosslinks to both the 16S and 23S molecules, the most pronounced being (1) an entirely A-site-specific crosslink from tRNA position 20:1 to the loop-end region (nt 877-913) of helix 38 of the 23S RNA (a region that has not so far been associated at all with tRNA binding), and (2) a largely P-site-specific crosslink from tRNA position 8 to nt 2111-2112 of the 23S RNA (nt 2112 being a position that has previously been identified in footprinting studies as belonging to the ribosomal E site). The data are compared with results from a parallel study of crosslinks from position 47 (also in the central fold of the tRNA), as well as with previously published crosslinks from the anticodon loop (positions 32, 34, and 37) and the CCA-end region (position 76, and the aminoacyl residue).

Getting closer to an understanding of the three-dimensional structure of ribosomal RNA.

Two experimentally unrelated approaches are converging to give a first low-resolution solution to the question of the three-dimensional organization of the ribosomal RNA from Escherichia coli. The first of these is the continued use of biochemical techniques, such as cross-linking, that provide information on the relative locations of different regions of the RNA. In particular, recent data identifying RNA regions that are juxtaposed to functional ligands such as mRNA or tRNA have been used to construct improved topographical models for the 16S and 23S RNA. The second approach is the application of high-resolution reconstruction techniques from electron micrographs of ribosomes in vitreous ice. These methods have reached a level of resolution at which individual helical elements of the ribosomal RNA begin to be discernible. The electron microscopic data are currently being used in our laboratory to refine the biochemically derived topographical RNA models.

Contacts between 16S ribosomal RNA and mRNA, within the spacer region separating the AUG initiator codon and the Shine-Dalgarno sequence; a site-directed cross-linking study.

mRNA analogues containing several 4-thiouridine (thio-U) residues at selected positions were prepared by T7-transcription. The spacer region between the Shine-Dalgarno sequence and the AUG codon consisted of four or eight bases with a single thio-U at a variable position; alternatively, cro-mRNA analogues were used carrying the thio-U substituted spacer sequence UUGU. The mRNAs were bound to E. coli ribosomes, and--after irradiation--the sites of cross-linking to 16S RNA were analysed. Three cross-links to the 16S RNA from the spacer region were observed, namely to positions 665, 1360, and a site close to nucleotide 1530. The cross-links were formed in different amounts in the presence or absence of tRNA(fMet), and were observed from thio-U residues located at various positions within the spacer sequence, although in the presence of tRNA they were in general stronger from positions close to the Shine-Dalgarno end of the spacer. The cross-linking behaviour in this upstream area of the mRNA is thus rather different in character from the previously published pattern in the downstream area. From considerations of structural conservation in small subunit RNA, we propose that both the upstream and downstream cross-links to 16S RNA reflect a universal mRNA path through the ribosome.

Stem-loop IV of 5S rRNA lies close to the peptidyltransferase center.

A DNA fragment containing the Escherichia coli 5S rDNA sequence linked to a T7 promoter was prepared by PCR from an M13 clone carrying the 5S-complementary sequence. The DNA was transcribed with T7 polymerase using a mixture of [alpha-32P]UTP and 4-thio-UTP, yielding a transcript in which approximately 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.

Site-directed cross-linking of mRNA analogues to 16S ribosomal RNA; a complete scan of cross-links from all positions between '+1' and '+16' on the mRNA, downstream from the decoding site.

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.

Three widely separated positions in the 16S RNA lie in or close to the ribosomal decoding region; a site-directed cross-linking study with mRNA analogues.

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.

The path of mRNA through the Escherichia coli ribosome; site-directed cross-linking of mRNA analogues carrying a photo-reactive label at various points 3' to the decoding site.

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.

The three-dimensional structure and function of Escherichia coli ribosomal RNA, as studied by cross-linking techniques.

A large number of intra-RNA and RNA-protein cross-link sites have been localized within the 23S RNA from E. coli 50 S ribosomal subunits. These sites, together with other data, are sufficient to constrain the secondary structure of the 23 S molecule into a compact three-dimensional shape. Some of the features of this structure are discussed, in particular, those relating to the orientation of tRNA on the 50 S subunit as studied by site-directed cross-linking techniques. A corresponding model for the 16S RNA within the 30 S subunit has already been described, and here a site-directed cross-linking approach is being used to determine the path followed through the subunit by messenger RNA.

Site-directed cross-linking of mRNA analogues to the Escherichia coli ribosome; identification of 30S ribosomal components that can be cross-linked to the mRNA at various points 5' with respect to the decoding site.

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.