Yeast tRNA-splicing endonuclease cleaves precursor tRNA in a random pathway.

Introns interrupt many of the tRNA genes of Saccharomyces cerevisiae at a constant position in the anticodon loop. Pre-tRNA transcripts must be accurately cleaved at 3' and 5' splice sites by tRNA endonuclease to release these introns. In order to study splice site cleavage order, substrates were prepared in which the ribose 2'-OH at each of the splice sites was phosphorylated. This modification blocked cleavage by the endonuclease. We found that whichever splice site was blocked the endonuclease can cleave the other site, indicating that the two splice sites were cleaved independently. The endonuclease also cleaved both 3'- and 5'-nicked pre-tRNA(Phe). In addition, both kinds of "2/3 molecules" (exon+intron) were observed in kinetic studies, indicating that they were true biochemical intermediates. The rates of cleavage at the 3' and 5' splice sites of pre-tRNA were compared in several ways. The results showed that the endonuclease cleaves 3' and 5' sites at almost the same rate in the first cleavage, whereas in the second cleavage the 3' site was cleaved faster, indicating that the rates of the two routes for cleavage were unequal. These results demonstrated that the endonuclease cleaved pre-tRNA in a random order, creating two routes for removal of introns from pre-tRNA.