Conservation of RNA sequence and crosslinking ability in ribosomes from a higher eukaryote: photochemical crosslinking of the anticodon of P site bound tRNA to the penultimate cytidine of the UACACCG sequence in Artemia salina 18S rRNA

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Abstract
The complex of Artemia salina ribosomes and Escherichia coli acetylvalyl-tRNA could be cross-linked by irradiation with near-UV light. Cross-linking required the presence of the codon GUU, GUA being ineffective. The acetylvalyl group could be released from the cross-linked tRNA by treatment with puromycin, demonstrating that cross-linking had occurred at the P site. This was true both for pGUU- and also for poly(U2,G)-dependent cross-linking. All of the cross-linking was to the 18S rRNA of the small ribosomal subunit. Photolysis of the cross-link at 254 nm occurred with the same kinetics as that for the known cyclobutane dimer between this tRNA and E. coli 16S rRNA. T1 RNase digestion of the cross-linked tRNA yielded an oligonucleotide larger in MW than any from un-cross-linked rRNA or tRNA or from a prephotolyzed complex. Extended electrophoresis showed this material to consist of two oligomers of similar mobility, a faster one-third component and a slower two-thirds component. Each oligomer yielded 2-components on 254-nm photolysis. The slower band from each was the tRNA T1 oligomer CACCUCCCUVACCAGp, which includes the anticodon. The faster band was the rRNA 9-mer UACACACCGp and its derivative UACACACUG. The dephosphorylated and slower moving 9-mer was derived from the faster moving dimer. Deamination of the penultimate C to U is probably due to cyclobutane dimer formation and was evidence for that nucleotide being the site of cross-linking. Direct confirmation of the cross-linking site was obtained by Z-gel analysis. Cross-linked tRNA nucleotide was the V base and that < 1% of the cross-linking could have involved the 5''-adjacent cytidine of the rRNA oligonucleotide. The cross-linking properties and sites of cross-linking are the same as those for E. coli and yeast ribosomes. The close contact between the middle of an almost completely conserved sequence loop of rRNA and the anticodon of tRNA must have considerable functional importance to be preserved over such a divergent range of species.

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