Nobel Centennial Symposia"Frontiers of Molecular Science"

December 4-7, 2001
Friiberghs Manor, Örsundsbro and Stockholm University

Functional Insights from the Structures of the Large Ribosomal Subunit and its Complexes with Ligands

by Steitz, T.A. (1,2,3), Hansen, J. (1), Schmeing, M. (1), Klein, D. (1), Ban, N. (1), Nissen, P. (1) and Moore, P.B. (2,1)
(1) Department of Molecular Biophysics & Biochemistry and (2) Department of Chemistry, Yale University and (3) Howard Hughes Medical Institute, New Haven, CT 06520-8114, USA

We have determined the crystal structure of the Haloarcula marismortui large ribosomal subunit at 2.4 Å resolution (1), and the structures of its complexes with substrate, product and intermediate analogues (2) as well as with antibiotics. The domains formed by its 3000 nucleotide of RNAs all have irregular shapes and fit together like the pieces of a three-dimensional jigsaw puzzle to form a large, monolithic structure. The 27 proteins are abundant everywhere on its surface except in the active site where peptide bond formation occurs and on the surface where the large subunit contacts the small subunit. Most of the proteins stabilize the structure by interacting with several RNA domains, often using idiosyncratically folded extensions that reach into the subunit's interior. Abundant examples of two previously unrecognized RNA motifs are seen. One, the A-minor motif (3), appears to be important in stabilizing RNA-RNA tertiary structure, and the other, the kink-turn (4), forms part of the binding site for several proteins and is likely to occur in many other ribonuclear protein complexes.

Using the structures of substrate and product complexes, we have established that only RNA is directly involved in the catalysis of peptidyl transferase by the ribosome since these analogs are contacted exclusively by conserved rRNA residues from domain V of 23S rRNA. The mechanism of peptide bond synthesis is hypothesized to involve the orientation of A-site and P-site substrates by 23 S rRNA and the participation of the base of A2486 (A2451 in E. coli), which contributes to catalysis by orienting A-site substrate and possibly by playing a general acid/base role. Addition of fragment substrates to the A and P sites in the crystal lead to the formation of products, as assayed both crystallographically and biochemically. Several antibiotics are observed to bind in the polypeptide exit tunnel, either overlapping with or near the substrate analogues. The macrolides function by blocking the egress of newly synthesized polypeptide.

New directions include establishing the structures of the 70S ribosome captured in its various functional states, including the secretion of proteins through the membrane bound translocon. Further, the development of novel antibiotics can now be approached using the methods of structure based drug design, a method used previously on macromolecules 100 times smaller.

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(1) Ban, N. Nissen, P., Hansen, J., Moore, P.B. and Steitz, T.A. The complete atomic structure of the large ribosomal subunit at 2.4 Å resolution. Science 289: 905-920 (2000).

(2) Nissen, P., Hansen, J., Ban, N., Moore, P.B. and Steitz, T.A. The structural basis of ribosome activity in peptide bond synthesis. Science 289: 920-930 (2000).

(3) Nissen, P., Ippolito, J.A., Ban, N., Moore, P.B. and Steitz, T.A. RNA tertiary interactions in the large ribosomal subunit: The A-minor motif. Proc. Natl. Acad. Sci. USA 98: 4899-4903 (2001).

(4) Klein, D.J., Schmeing, T.M., Moore, P.B. and Steitz, T.A. The kink-turn: a new RNA secondary structure motif. EMBO J. 20: 4214-4221 (2001).