Anticodon reconstruction of inosine-containing transfer RNA species

The mechanism for inosine biosynthesis in the tRNA anticodon wobble position (position 34) was recently established. An enzyme (a putative tRNA-hypoxanthine ribosyltransferase) inserts preformed hypoxanthine by an apparent base exchange reaction. Characterization of this enzyme requires an unmodified tRNA substrate containing adenosine in the wobble position. Following procedures similar to those described previously, they reconstructed yeast tRNA/supb Ala/ (anticodon IGC) to contain the anticodon AGC. Using S/sub 1/ and T/sub 1/ nucleases under mild conditions, the tRNA molecule was cleaved after U/sub 33/ and G/sub 35/ respectively. The tRNA half molecules, lacking residues 34 and 35, were first separated, then reannealed. The dinucleotide AG, with a 5' /sup 32/P label, was then ligated into the gap to yield an unmodified (A/sub 34/) alanine tRNA. This tRNA (while not a substrate for inosine modification in Xenopus oocytes) is being evaluated as a substrate in vitro for the human cell modification enzyme. Other tRNAs are being similarly reconstructed, and the effects of modifications at position 37 (e.g., 1-methylinosine) are being evaluated.