Interaction of elongation factors EF-G and EF-Tu with a conserved loop in 23S RNA

Abstract

The elongation factors EF-Tu and EF-G interact with ribosomes during protein synthesis^1,2: EF-Tu presents incoming aminoacyl transfer RNA to the programmed ribosome as an EF-Tu-GTP-tRNA ternary complex and EF-G promotes translocation of peptidyl-tRNA and its associated messenger RNA from the A to the P site after peptidyl transfer. Both events are accompanied by ribosome-dependent GTP hydrolysis. Here we use chemical probes to investigate the possible interaction of these factors with ribosomal RNA in E. coli ribosomes. We observe EF-G-dependent footprints in vitro and in vivo around position 1,067 in domain II of 23S rRNA, and in the loop around position 2,660 in domain VI. EF-Tu gives an overlapping footprint in vitro at positions 2,655 and 2,661, but shows no effect at position 1,067. The 1,067 region is the site of interaction of the antibiotic thiostrepton², which prevents formation of the EF-G–GTP–ribosome complex and is a site for interaction with the GTPase-related protein L11 (ref. 3). The universally conserved loop in the 2,660 region⁴ is the site of attack by the RNA-directed cytotoxins α-sarcin⁵ and ricin⁶, whose effects abolish translation and include the loss of elongation factor-dependent functions⁷ in eukaryotic ribosomes.