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Albany 2001

category image Biomolecular
Stereodynamics
SUNY at Albany
June 19-23, 2001

Functional Complexes of the 70S Ribosome

Crystal structures of Thermus thermophilus 70S ribosomes complexed with mRNA and two or three tRNAs have been solved to resolutions of up to 5.5 A (1,2). At this resolution, the chains of the 16S, 5S and 23S rRNAs can be traced completely, and all of the ribosomal proteins of known structure can be fitted to the electron density. Significant differences can be seen in the structures of the 30S and 50S subunits in the 70S ribosome, compared with the high-resolution structures of the free subunits (3-5).

The molecular interactions in all of the twelve intersubunit bridges have been identified. All three possible types of interaction - RNA-RNA, RNA-protein and protein-protein - are observed. The core bridge interactions around the center of the interface, close to the tRNA binding sites, are mainly RNA-RNA interactions, while those involving proteins tend to be distributed around the periphery of the interface. RNA-RNA bridge contacts are dominated by minor groove-minor groove interactions between RNA helices from 16S and 23S rRNA.

The structure of the P-tRNA can be fitted directly to the 5.5 A map with little change in its structure compared with the structure of free tRNA, while the E-tRNA is noticeably distorted in several places by its interactions with the ribosome. Significant interactions are made between E-tRNA and the ribosome in the 30S subunit as well as the 50S subunit, although its anticodon appears to interact only minimally with the mRNA. The A-tRNA was positioned in a 7 A Fourier difference map; negative density is observed at the positions of bases A1492 and 1493, providing evidence for their proposed flipping into the minor groove of the A-codon-anticodon helix (6). In the absence of A-tRNA, protein density overlaps its 50S binding site, indicating that a conformational change in a 50S ribosomal protein (most likely L16) must take place during the accommodation step of A-tRNA binding. The main interactions between the tRNAs and the ribosome are with 16S and 23S rRNA, but all three tRNAs also interact with ribosomal proteins.

The path of the mRNA through the 30S subunit was visualized directly by Fourier difference maps using ribosomal complexes programmed with three different mRNAs and complexes in which all components except mRNA were present. The part of the mRNA bound by the ribosome comprises 30 +/-1 nucleotides, within experimental error of the value reported by nuclease protection studies more than thirty years ago (7).

Some of the intersubunit bridges are located very close to, or even in contact with the tRNAs. There is evidence from several different approaches that these particular bridges are dynamic structural elements of the ribosome. The implication that they may participate in tRNA translocation is consistent with the idea that translocation involves dynamic interactions in the ribosome structure at the subunit interface.

References and Footnotes
  1. Cate JH, Yusupov MM, Yusupova GZ, Earnest TN, and Noller HF (1999) Science 285:2095-2104.
  2. Yusupov, M.M., Yusupova, G.Zh., Baucom, A., Lieberman, K., Earnest, T.N., Cate, J.H.D. and Noller, H.F. (2001) submitted
  3. Ban N, Nissen P, Hansen J, Moore PB and Steitz TA (2000) Science 289:905-920.
  4. Wimberly BT, Brodersen DE, Clemons WM, Morgan-Warren RJ, Carter AP, Vonrhein C, Hartsch T and Ramakrishnan V (2000) Nature 407:327-339.
  5. Schluenzen F, Tocilj A, Zarivach R, Harms J, Gluehmann M, Janell D, Bashan A, Bartels H, Agmon I, Franceschi F and Yonath A (2000) Cell 102:615-623.
  6. Carter AP, Clemons WM, Brodersen DE, Morgan-Warren RJ, Wimberly BT and Ramakrishnan V (2000) Nature 407:340-348
  7. Steitz JA (1969) Nature 224:957-964

Harry F. Noller(1), Marat M. Yusupov(1,2), Gulnara Zh. Yusupova(1,2), Albion Baucom(1), Kate Lieberman(1), Thomas N. Earnest(3), Laura Lancaster(1), Anne Dallas(1), Kurt Fredrick(1), J.H.C. Cate(4)

Center for Molecular Biology of RNA(1), UCSC, Santa Cruz, CA 95064; Present Address(2): Laboratoire de Biologie et Genomique Structurales de l'IGBMC, CNRS, Strasbourg France; (3) Macromolecular Crystallography Facility, Advanced Light Source, LBNL, Berkeley, CA 94720; (4) Whitehead Institute, MIT, Cambridge MA 01242.