TRUNCATED ELONGATION-FACTOR-G LACKING THE G DOMAIN PROMOTES TRANSLOCATION OF THE 3'-END BUT NOT OF THE ANTICODON DOMAIN IN OF PEPTIDYL-TRANSFER-RNA

The mechanism by which elongation factor G (EF-G) catalyzes the transl ocation of tRNAs and mRNA on the ribosome is not known. The reaction r equires GTP, which is hydrolyzed to GDP. Here we show that EF-G from E scherichia coli lacking the G domain still catalyzed partial transloca tion in that it promoted the transfer of the 3' end of peptidyl-tRNA t o the P site on the 50S ribosomal subunit into a puromycin-reactive st ate in a slow-turnover reaction. In contrast, it did not bring about t ranslocation on the 30S subunit, since (i) deacylated tRNA was not rel eased from the P site and (ii) the A site remained blocked for aminoac yl-tRNA binding during and after partial translocation. The reaction p robably represents the first EF-G-dependent step of translocation that follows the spontaneous formation of the A/P state that Is not puromy cin-reactive [Moazed, D. & Noller, H. F. (1989) Nature (London) 342, 1 42-148]. In the complete system-i.e., with intact EF-G and GTP-the 50S phase of translocation is rapidly followed by the 30S phase during wh ich the tRNAs together with the mRNA are shifted on the small ribosoma l subunit, and GTP is hydrolyzed. As to the mechanism of EF-G function , the results show that the G domain has an important role, presumably exerted through interactions with other domains of EP-G, in the promo tion of translocation on the small ribosomal subunit. The G domain's i ntramolecular interactions are likely to be modulated by GTP binding a nd hydrolysis.