Book of Abstracts: Albany 2003
June 17-21 2003
Travelling with t-RNAs Through the Ribosome
The transfer-RNA (tRNA) in its 20 varieties is the quintessential tool used by the ribosome in protein biosynthesis. The three binding sites of tRNA (A, P, and E) have been localized on the ribosome both by cryo-electron microscopy (cryo-EM) (1) and x-ray crystallography (2). During translation, translocation of tRNA and mRNA through ribosome occurs in a coordinated fashion. We have earlier found that the movement of tRNA is accompanied by large-scale rearrangements in the ribosome (3).
Most recently, relatively high-resolution cryo-EM maps of 70S ribosome, in the range of 9-12 A, have become available for sequential functional states during translation. These functional states of ribosome include (i) an initiation-like state, (ii) the ribosome bound with aa-tRNA.EF-Tu.GDP (ternary complex), (iii) the ribosome with tRNA accommodated in the A site, (iv) the state after peptide transfer ("pre-translocational state"), and (v) after EF-G interaction ("post-translocational state"). Apart from those, the ribosome bound with EF-G with tRNA in the hybrid (P/E) state, and with paromomycin-stalled A-site tRNA were also reconstructed. In all these maps, tRNAs are directly recognizable in the inter-subunit space of the 70S.
Localization of tRNAs in each of these functional states reveals the path of the tRNAs through the ribosome. A real-space refinement technique has been applied (4) to construct atomic models of the dynamically changing ribosome, from which the detailed interactions with the tRNA can be inferred.
1Howard Hughes Medical Institute