Richard Brimacombe

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 Å (refs 2, 3) 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 ∼20 Å. Our three-dimensional reconstruction at 18 Å 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 70S Escherichia coli ribosome at 23 å resolution: fitting the ribosomal RNA

BACKGROUND The ribosome--essential for protein synthesis in all organisms--has been an evasive target for structural studies. The best available structures for the 70S Escherichia coli ribosome or its 30S and 50S subunits are based on electron microscopical tilt experiments and are limited in resolution to 28-55 A. The angular reconstitution approach, which exploits the random orientations of particles within a vitreous ice matrix, can be used in conjunction with cryo-electron microscopy to yield a higher-resolution structure. RESULTS Our 23 A resolution map of the 70S ribosome elucidates many structural details, such as an extensive system of channels within the 50S subunit and an intersubunit gap ideally shaped to accommodate two transfer RNA molecules. The resolution achieved is sufficient to allow the preliminary fitting of double-helical regions of an earlier three-dimensional ribosomal RNA model. CONCLUSIONS Although we are still a long way from attaining an atomic-resolution structure of the ribosome, cryo-electron microscopy, in combination with angular reconstitution, is likely to yield three-dimensional maps with gradually increasing resolution. As exemplified by our current 23 A reconstruction, these maps will lead to progressive refinement of models of the ribosomal RNA.

Clustering of modified nucleotides at the functional center of bacterial ribosomal RNA.

An aryl trifluoromethyl diazirine photo‐reactive derivative was attached to the 2‐thiocytidine residue at position 32 of tRNAIArg and this derivatized tRNA was bound to Escherichia coli 70S ribosomes. After irradiation at 350 nm the site of cross‐linking to the 16S RNA was analyzed by our standard procedures and found to lie within the secondary structural element comprising bases 956‐983; this region contains two modified nucleotides at positions 966 and 967. Similarly, an aryl azido photoreactive derivative was attached to the phenylalanine residue of Phe‐tRNAPhe, and the derivatized aminoacyl tRNA was bound to the ribosome either at the A‐ or the P‐site. In both cases, after irradiation at 250 nm, the cross‐link site was localized to position 2439 of the 23S RNA; in the secondary structure of the latter the neighboring nucleotide 2442 is base‐paired to a modified nucleotide at position 2069. Taken together with other cross‐linking data, these results now directly implicate a total of 27 out of the 29 modified nucleotides in E. coli 16S and 23S RNA as lying within or close to the functional center of the ribosome.— Brimacombe, R., Mitchell, P., Osswald, M., Stade, K., Bochkariov, D. Clustering of modified nucleotides at the functional center of bacterial ribosomal RNA. FASEB J. 7: 161‐167; 1993.

The decoding region of 16S RNA; a cross-linking study of the ribosomal A, P and E sites using tRNA derivatized at position 32 in the anticodon loop.

A photo‐reactive diazirine derivative was attached to the 2‐thiocytidine residue at position 32 of tRNA(Arg)I from Escherichia coli. This modified tRNA was bound under suitable conditions to the A, P or E site of E.coli ribosomes. After photo‐activation of the diazirine label, the sites of cross‐linking to 16S rRNA were identified by our standard procedures. Each of the three tRNA binding sites showed a characteristic pattern of cross‐linking. From tRNA at the A site, a major cross‐link was observed to position 1378 of the 16S RNA, and a minor one to position 936. From the P site, there were major cross‐links to positions 693 and to 957 and/or 966, as well as a minor cross‐link to position 1338. The E site bound tRNA showed major cross‐links to position 693 (identical to that from the P site) and to positions 1376/1378 (similar, but not identical, to the cross‐link observed from the A site). Immunological analysis of the concomitantly cross‐linked ribosomal proteins indicated that S7 was the major target of cross‐linking from all three tRNA sites, with S11 as a minor product. The results are discussed in terms of the overall topography of the decoding region of the 30S ribosomal subunit.

The location of mRNA in the ribosomal 30S initiation complex; site-directed cross-linking of mRNA analogues carrying several photo-reactive labels simultaneously on either side of the AUG start codon.

Messenger RNA molecules 30–35 bases long, with sequences related to the 5′‐region of cro‐mRNA from lambda‐phage, were prepared by T7 transcription from synthetic DNA templates. Each mRNA contained five or six internal uridine residues, which were transcribed using a mixture of UTP and thio‐UTP. Initiation complexes were formed with Escherichia coli 30S ribosomes in the presence or absence of tRNA(fMet), and cross‐linking of the thio‐U residues was induced by UV irradiation at wavelengths greater than 300 nm. The cross‐linked ribosomal proteins were identified immunologically, and cross‐linked regions of the 16S RNA were isolated by excision with ribonuclease H and suitable deoxyoligonucleotides. In both cases, the particular thio‐U residue involved in the cross‐link was identified by ribonuclease T1 fingerprinting of the (radioactive) mRNA in the isolated cross‐linked complex. The principal results were that, at thio‐U positions upstream of the AUG codon, specific cross‐linking occurred to protein S7 and to the 3′‐terminus of the 16S RNA, in agreement with similar experiments using 70S ribosomes. Less specific cross‐linking was observed to proteins S1, S18 and S21 at various positions within the mRNA. Six bases downstream from the AUG codon, a tRNA‐dependent cross‐link was found to position approximately 1050 of the 16S RNA, but‐‐in contrast to similar experiments with 70S ribosomes‐‐no cross‐linking was found to the 1390–1400 region.

Specific cross-linking of proteins S7 and L4 to ribosomal RNA, by UV irradiation of Escherichia coli ribosomal subunits

SummaryRadioactive 30S and 50S subunits from E. coli ribosomes were irradiated with UV light, under conditions giving rise to approximately 10% cross-linking of protein to ribosomal RNA. Irradiation to levels of cross-linking higher than 10% caused unfolding of the ribosomal subunits, even in the presence of 5 mM magnesium.The specificity of the cross-linking reaction at this low level was found to be extremely high. Cross-linked RNA-protein complexes, freed from unbound protein, were treated with nuclease and then analysed on Sarkosyl gels. S7 was found to be the primary target of the cross-linking reaction in the 30S particle. This was proven by using subunits from both E. coli MRE 600 and A19, whose respective S7 species differ markedly. In the 50S particle, L4 was the primary target, although L2 was also cross-linked to a small extent. Ambiguity in the identification of L4 in the Sarkosyl system was resolved by two-dimensional electrophoresis, which was also used to demonstrate a genuine linkage to RNA in the case of both S7 and L4; protein spots containing 32P were observed, derived from these two proteins, when subunits containing 32P-RNA were irradiated, treated with nuclease, and applied to the electrophoresis. The identities of S7, L4 and L2 were finally confirmed by Ouchterlony tests with protein-specific anti-sera.

A model for the spatial arrangement of the proteins in the large subunit of the Escherichia coli ribosome.

A three‐dimensional model for the arrangement of 29 of the 33 proteins from the Escherichia coli large ribosomal subunit has been generated by interactive computer graphics. The topographical information that served as input in the model building process was obtained by combining the immunoelectron microscopically determined network of epitope‐epitope distances on the surface of the large ribosomal subunit with in situ protein‐protein cross‐linking data. These two independent sets of data were shown to be compatible by geometric analysis, thus allowing the construction of an inherently consistent model. The model shows (i) that the lower third of the large subunit is protein‐poor, (ii) that proteins known to be functionally involved in peptide bond formation and translocation are clustered in two separate regions, (iii) that proteins functionally interdependent during the self‐assembly of the large subunit are close neighbours in the mature subunit and (iv) that proteins forming the early assembly nucleus are grouped together in a distinct region at the ‘back’ of the subunit.

The Structure of Ribosomal RNA and Its Organization Relative to Ribosomal Protein

Publisher Summary The chapter describes how the sequence information has been used to derive convincing secondary structure models for the RNA from both subunits of the E. coli ribosome, and compares the various models that are proposed. It shows how extrapolation of these data to ribosomal RNA molecules of widely differing size classes leads to the clear conclusion that the secondary structures, as well as significant regions in the primary sequences, have been conserved to a large extent throughout evolution. The chapter deals with the three dimensional organization of the ribosomal RNA and its arrangement with respect to the ribosomal proteins, concentrating once again on the E. coli ribosome. In particular, it includes a review of the application of cross-linking techniques (bath RNA to protein and intra-RNA) to this problem. In general, rather than presenting an exhaustive survey of the literature, the chapter has selected topics or examples to illustrate those problems or points of interest that one considers to be most relevant to the central objective in this field of research— namely, the elucidation of the three-dimensional structure of the ribosomal RNA in situ 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.

Identification of the oligonucleotide and oligopeptide involved in an RNA-protein crosslink induced by ultraviolet irradiation of Escherichia coli 30 S ribosomal subunits

Abstract When 30 S ribosomal subunits are irradiated with ultraviolet light, we have found that an RNA-protein crosslinking reaction occurs whose primary target is protein S7. This paper describes the identification of the oligopeptide and oligonucleotide at the crosslinking point, by direct analysis (a) of the peptide remaining attached to an oligonucleotide (after total digestion of the RNA in the crosslinked complex with ribonucleases A and T1, followed by digestion with trypsin), and (b) of the nucleotides remaining attached to the crosslinked protein (after digestion of the RNA in the complex with ribonuclease T1 alone). The crosslinking site was found to lie within a single short peptide, Ser-Met-Ala-Leu-Arg (positions 113 to 117 in the S7 sequence), with methionine as the probable amino acid concerned. The principal RNA site was found to lie within an oligonucleotide three to six bases long, the underlined portion of the partially ordered sequence C-U-A-C- A-A-U-G.G.C -G in section P of the 16 S RNA. The methodology involved has been designed with a view to being generally applicable in future RNA-protein crosslinking studies, where several proteins are simultaneously attached to the RNA.