MECHANISM, SPECIFICITY AND GENERAL-PROPERTIES OF THE YEAST ENZYME CATALYZING THE FORMATION OF INOSINE-34 IN THE ANTICODON OF TRANSFER-RNA

In yeast, inosine is found at the first position of the anticodon (pos ition 34) of seven different isoacceptor tRNA species, while in Escher ichia coli it is present only in tRNA(Arg). The corresponding tRNA gen es all have adenosine at position 34. Using as substrates in vitro T7- runoff transcripts of 31 plasmids carrying each natural of synthetic t RNA gene harbouring an anticodon with adenosine 34, we have characteri sed a yeast enzyme that catalyses the conversion of adenosine 34 to in osine 34. The homologous E. coli enzyme modifies adenosine 34 only in tRNAs with an arginine anticodon ACG. The base conversion occurs by a hydrolytic deamination-type reaction. This was determined by reversed phase high-pressure liquid chromatography/electrospray mass spectromet ry analysis of the reaction product after in vitro modification in [O- 18]water. This newly characterised tRNA:adenosine 34 deaminase was par tially purified from yeast. It has a molecular mass of approximately 7 5 kDa, and it does not require any cofactor, except magnesium ions, to deaminate adenosine 34 efficiently in tRNA. The observed dependence o f the enzymatic reaction on magnesium ions probably reflects the need for a correct tRNA architecture. Enzymatic recognition of tRNA does no t depend on the presence of any ''identity'' nucleoside other than ade nosine 34. Likewise, the presence of pseudouridine 32 or 1-methyl-guan osine 37 in the anticodon loop does not interfere with inosine 34 bios ynthesis. However, the efficacy of adenosine 34 to inosine 34 conversi on depends on the nucleotide sequence of the anticodon loop and its pr oximal stem, the best tRNA substrates being those with a purine at pos ition 35. Mutations that affect the size of the anticodon loop or one of several three-dimensional base-pairs abolish the capacity of the tR NA to be substrate for the yeast tRNA:adenosine 34 deaminase. Evidentl y, the activity of yeast tRNA:adenosine 34 deaminase depends more on t he global structural feature (conformational stability/flexibility) of the L-shaped tRNA substrates than on the identity of any particular n ucleotide other than adenosine 34. An apparent K-m of 2.3 nM for its n atural substrate tRNA(Ser) (anticodon AGA) was measured. Altogether, t hese results suggest that a single enzyme can account for the presence of inosine 34 in all seven cytoplasmic A34-containing precursor tRNAs in yeast. (C) 1996 Academic Press Limited