Introduction

This group has for many years been concerned with the study of the structure of the ribosomal RNA (rRNA) molecules from the eubacterium Escherichia coli. After deriving secondary structure models for both the small (16S) subunit rRNA (ref. 2) and the corresponding 23S rRNA from the large subunit (ref. 1), we turned our attention to the three-dimensional structure of these molecules, making use of cross-linking techniques. Both intra-RNA (e.g. ref. 3, 4) and RNA-protein (e.g. ref. 5, 8) cross-linking methods were applied, so as to gain information describing contacts and neighbourhoods between different regions of the rRNA molecules themselves, as well as between the rRNA and individual ribosomal proteins. Particular emphasis was laid on developing methods for the precise analysis of cross-link sites within the rRNA. The result of these studies was a first three-dimensional model for the 16S rRNA (ref. 6), and a preliminary model for the 23S rRNA (ref. 7).

We then began to develop more sophisticated "site-directed" cross-linking techniques, so as to study the interaction of functional ligands such as mRNA or tRNA with the ribosome. In this method, a photo-reactive group is introduced at a chosen position within the ligand under investigation, and - after binding to the ribosome and appropriate photo-activation - the cross-link sites to the ribosomal conponents are identified. This approach, in collaboration with the group of Alexey Bogdanov and Olga Dontsova (Moscow State University) proved particulary succesful with mRNA (e.g. ref. 9), and led to the identification of a set of highly specific cross-links between mRNA and sites on the 16S rRNA. Corresponding experiments with tRNA (e.g. ref. 13), using for the most part the diazirine cross-linking reagents developed in Russia, also yielded a great deal of new information. The data concerned are reviewed in ref. 10. More recently, similar techniques have been applied to the study of the environment of the N-terminus of the growing peptide chain (refs. 11, 21) and of the 5S rRNA (refs. 14, 23) within the large ribosomal subunit. A technique has also been developed for the insertion of a photo-reactive label at selected sites in the 16S or 23S rRNA molecules, which has given us new information on their three-dimensional folding (refs. 16, 22)

The most dramatic development with regard to the three-dimensional folding of the rRNA molecules has however come from a completely different direction, namely from cryo-electron microscopy. The application of this method (in which the specimen is rapidly frozen in vitreous ice), combined with new techniques for computerized image reconstruction, has resulted in the derivation of structures of the E.coli ribosome at increasingly high resolution (ref. 12). At this level of resolution, the ribosomally bound tRNA molecules can be directly seen (ref. 15), as well as ligands such as elongation factor Tu (ref. 17). Fine structural elements about 20 Å in diameter, which clearly correspond to helical elements of the rRNA, are also visible in these structures. The current level of resolution from the laboratory of Holger Stark and Marin van Heel (Imperial College, London) has reached 13 Å, and the 16S and 23S rRNA molecules are in the process of being fitted to the 13 Å reconstruction. The 16S rRNA had already been fitted to a reconstruction at somewhat lower resolution (refs. 18, 19, 20), in a manner which satisfies the great majority of the biochemical facts.

The actual fitting of the molecules is done mostly interactively using graphics workstations. For this purpose we have developped methods for the visualization and manipulation of large RNA molecules. For our current modelling studies we use the programme ERNA-3D, which allows the selective display and dynamic manipulation of the rRNA.


References

1. Glotz, C., Zwieb, C., Brimacombe, R., Edwards, K. and Kössel, H. (1981). Secondary structure of the large subunit ribosomal RNA from E.coli, Z. mays chloroplast, and human and mouse mitochondrial ribosomes. Nucleic Acids Res. 9, 3287-3306.

2. Zwieb, C., Glotz, C. and Brimacombe, R. (1981). Secondary structure comparisons between small subunit ribosomal RNA molecules from six different species. Nucleic Acids Res. 9, 3621-3640.

3. Atmadja, J., Stiege, W., Zobawa, M., Greuer, B., Oßwald, M. and Brimacombe, R. (1986). The tertiary folding of E.coli 16S RNA, as studied by in situ intra-RNA cross-linking of 30S ribosomal subunits with bis-(2-chloroethyl)-methylamine. Nucleic Acids Res. 14, 659-673.

4. Stiege, W., Atmadja, J., Zobawa, M. and Brimacombe, R. (1986). Investigation of the tertiary folding of E.coli ribosomal RNA by intra-RNA cross-linking in vivo. J. Mol. Biol. 191, 135-138.

5. Oßwald, M., Greuer, B., Brimacombe, R., Stöffler, G., Bäumert, H. and Fasold, H. (1987). RNA-protein cross-linking in E.coli 30S ribosomal subunits; determination of sites on 16S RNA that are cross-linked to proteins S3, S4, S5, S7, S8, S9, S11, S13, S19 and S21 by treatment with p-azidophenyl acetimidate. Nucleic Acids Res. 15, 3221-3240.

6. Brimacombe, R., Atmadja, J., Stiege, W. and Schüler, D. (1988). A detailed model of the three-dimensional structure of E.coli 16S ribosomal RNA in situ in the 30S subunit. J. Mol. Biol. 199, 115-136.

7. Mitchell, P., Oßwald, M., Schüler, D. and Brimacombe, R. (1990). Selective isolation and detailed analysis of intra-RNA cross-links induced in the large ribosomal subunit of E.coli ; a model for the tertiary structure of the tRNA binding domain in 23S RNA. Nucleic Acids Res. 18, 4325-4333.

8. Oßwald, M., Greuer, B. and Brimacombe, R. (1990). Localization of a series of RNA-protein cross-link sites in the 23S and 5S ribosomal RNA from E.coli, induced by treatment of 50S subunits with three different bifunctional reagents. Nucleic Acids Res. 18, 6755-6760.

9. Dontsova, O., Dokudovskaya, S., Kopylov, A., Bogdanov, A. A., Rinke-Appel, J., Jünke, N. and Brimacombe, R. (1992). 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. EMBO J. 11, 3105-3116.

10. Brimacombe, R. (1995). The structure of ribosomal RNA; a three dimensional jigsaw puzzle. Eur. J. Biochem. 230, 365-383.

11. Stade, K., Jünke, N. and Brimacombe, R. (1995). Mapping the path of the nascent peptide chain through the 23S RNA in the 50S ribosomal subunit. Nucleic Acids Res. 23, 2371-2380.

12. Stark, H., Mueller, F., Orlova, E. V., Schatz, M., Dube, P., Erdemir, T., Zemlin, F., Brimacombe, R. and van Heel, M. (1995). The 70S ribosome at 23 Å resolution; fitting the ribosomal RNA. Structure 3, 815-821.

13. Rinke-Appel, J., Jünke, N., Oßwald, M. and Brimacombe, R. (1995). The ribosomal environment of tRNA; cross-links to rRNA from positions 8 and 20:1 in the central fold of tRNA located at the A, P or E site. RNA 1, 1018-1028.

14. Dokudovskaya, S., Dontsova, O., Shpanchenko, O., Bogdanov, A. and Brimacombe, R. (1996). Loop IV of 5S ribosomal RNA has contacts both to domain II and to domain V of the 23S RNA. RNA 2, 146-152.

15. Stark, H., Orlova, E. V., Rinke-Appel, J., Jünke, N., Mueller, F., Rodnina, M., Wintermeyer, W., Brimacombe, R. and van Heel, M. (1997). Arrangement of tRNAs in pre- and post-translocational ribosomes revealed by electron cryomicroscopy. Cell 88, 19-28.

16. Baranov, P. V., Dokudovskaya, S. S., Oretskaya, T. S., Dontsova, O. A., Bogdanov, A. A. and Brimacombe, R. (1997). A new technique for the characterization of long-range tertiary contacts in large RNA molecules: insertion of a photolabel at a selected position in 16S rRNA within the E.coli ribosome. Nucleic Acids Res., 25, 2266-2273.

17. Stark, H., Rodnina, M. V., Rinke-Appel, J., Brimacombe, R., Wintermeyer, W. and van Heel, M. (1997). Visualization of elongation factor Tu on the E.coli ribosome. Nature, 389, 403-406.

18. Mueller, F. and Brimacombe, R. (1997). A new model for the three-dimensional folding of E.coli 16S ribosomal RNA: I. Fitting the RNA to a 3D electron microscopic map at 20 Å. J. Mol. Biol., 271, 524-544.

19. Mueller, F. and Brimacombe, R. (1997). A new model for the three-dimensional folding of E.coli 16S ribosomal RNA: II. The RNA-protein interaction data. J. Mol. Biol., 271, 545-565.

20. Mueller, F., Stark, H., van Heel, M., Rinke-Appel, J. and Brimacombe, R. (1997). A new model for the three-dimensional folding of E. coli 16S ribosomal RNA: III. The topography of the functional centre. J. Mol. Biol., 271, 566-587.

21. Choi, K.M. and Brimacombe, R. (1998). The path of the growing peptide chain through the 23S rRNA in the 50S ribosomal subunit; a comparative cross-linking study with three different peptide families. Nulceic Acids Res. 26, 887-895.

22. Baranov, P.V., Gurvich, O.L., Bogdanov, A.A. Brimacombe, R. and Dontsova, O.A. (1998). New features of 23S ribosomal RNA folding: the long helix 41-42 makes a "U-turn" inside the ribosome. RNA, 4, 658-668.

23. Sergiev, P., Dokudovskaya, S., Romanova, E., Topin, A., Bogdanov, A., Brimacombe, R. and Dontsova, O. (1998). The environment of 5S rRNA in the ribosome: cross-links to the GTPase-associated area of the 23S rRNA. Nucleic Acids Res., 26, 2519-2525