Chapter 5 Site Directed Replacement of Nucleotides in the Anti-Codon Loop of Trna: Application to the Study of Inosine Biosynthesis in Yeast TRNAALA

Publisher Summary This chapter describes recombinant RNA technology which allows generating the required transfer RNA (tRNA) substrates in vitro by replacing specific nucleotides within the anticodon loop. Yeast tRNA Ala with the anticodon IGC was reconstructed to contain the anticodon AGC, and this tRNA was used as a substrate for the A 34 to I 34 modification catalyzed in vitro by the tRNA-hypoxanthine ribosyltransferase from cultured human promyelocytic leukemia cells. The key to utilizing this technology is based on placing a [ 32 P] label adjacent to the nucleoside of interest, so the modification reaction can be monitored. Recombinant RNAs are investigated by employing reconstructed tRNAs to examine specific modification reactions in vitro and in vivo. The site directed replacement of one or more nucleotides in pure tRNA isoacceptors has led to new insights into the mechanism and specificity of several modification enzymes acting at positions 34 and 37 in the anticodon loop, and similar technology should prove useful for future analyses of the nature and role of these important modifications.