Functional conservation near the 3' end of eukaryotic small subunit RNA: photochemical crosslinking of P site-bound acetylvalyl-tRNA to 18S RNA of yeast ribosomes

Abstract

Escherichia coli acetylvalyl (AcVal)-tRNA1Val became crosslinked to both yeast and spinach chloroplast ribosomes upon irradiation (300 nm) in the presence of poly(U2,G). Yields were 25-30% and 33%, respectively, compared to 45% for E. coli. Crosslinking occurred to the P site, only to the 40S subunit, and 90% of that was to the 18S rRNA. The crosslink could be photolyzed at 254 nm with the same first-order kinetics as for the E. coli ribosome complex previously studied. The AcVal-tRNA that split off could be crosslinked again when irradiated at 300 nm, showing that the crosslink was photoreversible. There was a strong codon specificity for crosslinking. With pG-U-U, 85% crosslinking was obtained after 20 min of irradiation; with G-U-A, only 3% crosslinking occurred. All of these properties are the same as those previously reported for the E. coli ribosome crosslink that occurs via cyclobutane dimer formation between the 5' anticodon base 5'-carboxymethoxyuridine-34 and cytidine-1400 of the 16S RNA. Cytidine-1400 is in the center of a 17-mer that has been almost totally conserved among the small subunit rRNAs of all species so far examined, including yeast. Crosslinking of tRNA in the same way to both yeast and E. coli ribosomes shows that there has been a functional conservation as well in this region of the small subunit rRNA. This region may be involved in some essential aspect of the decoding process that is common to both prokaryotic and eukaryotic protein synthesis systems.