|Budget Amount *help
¥2,500,000 (Direct Cost : ¥2,500,000)
Fiscal Year 1997 : ¥800,000 (Direct Cost : ¥800,000)
Fiscal Year 1996 : ¥1,700,000 (Direct Cost : ¥1,700,000)
The recognition of tRNA^<Leu>, one of the class IItRNAs having a long variable arm, by leucyl-tRNA synthetase in Saccharomyces cerevisiae was studied using the T7 transcription system. Exchanging the anticodon arm of tRNA^<Leu> but not the D-or TYC-arm to that of tRNA^<Ser> seriously affected the leucine accepting activity. Two nucleotides in the anticodon loop, A35 and G37, were foung to be important for leucylation. It was also found that the discriminator base, A37, is required for leucylation, and G37 of tRNA^<Ser> function as a negative identity determinant for leucyl-tRNA synthetase. Introducing a set of three base substitutions at 35,37 and 73 was sufficient to convert tRNA^<Ser> into an efficient leucine acceptor. These results indicate that the identity elements of tRNA^<Leu> lie at the second position of the anticodon and the 3' adjacent to the anticodon as well as the discriminator position. Such a sequence spedific recognition manner is significantly different from that of
Escherichia coli, in which not the anticodon but the tertiaty structural elements play a key role in discriminating from other class II tRNAs. The leucine system is the first example which shows that the requirement of the anticodon sequence is variable among species.
It was also found the unilateral aminoacylation specificity between Escherichia coli and Yeast Saccharomyces cerevisiae. Both SerRS and LeuRS from E.coli were unable to aminoacylate yeast class II tRNAs, in contrast, the yeast counterparts were able to aminoacylate E.coli class II tRNAs. Yeast SerRS was able to aminoacylate not only E.coli tRNA^<Ser> but also tRNA^<Leu> and tRNA^<Tyr>, and yeast LeuRS was able to aminoacylate not only E.coli tRNA^<Leu> but also tRNA^<Tyr>. These results indicate that the recognition style of class II tRNA is significantly divergent between E.coli and yeast, and also that the discrimination against noncognate tRNAs in yeastis less exclusive than that in E.coli. This difference is considered to be attributed considerably to the different composition of the class II tRNAs between E.coli and yeast.