No abstract

Accuracy in transfer RNA (tRNA) splicing is essential for the formation of functional tRNAs, and hence for gene expression, in both Eukaryotes and Archaea. The specificity for recognition of the tRNA precursor (pre-tRNA) resides in the endonuclease, which removes the intron by making two independent endonucleolytic cleavages. Although the eukaryal and archaeal enzymes appear to use different features of pre-tRNAs to determine the sites of cleavage, analysis of hybrid pre-tRNA substrates containing eukaryal and archaeal sequences, described here, reveals that the eukaryal enzyme retains the ability to use the archaeal recognition signals. This result indicates that there may be a common ancestral mechanism for recognition of pre-tRNA by proteins.

The tRNA splicing endonuclease cleaves intron-containing tRNA precursors on both sides of the intron. The prevailing belief has been that the enzyme binds only to the mature domain through the invariant bases. We show instead that, for recognition, the endonuclease utilizes distinct sets of structural elements, several of which are within the intron. One subset of recognition elements, localized in the mature domain, is needed for recognition of both cleavage sites, while two other subsets, localized at the exon-intron boundaries, are used for recognition of either one or the other cleavage site. The two cleavage sites are essentially independent: neither is required by the other for cleavage to take place. These results support a two-active-site model for the eucaryal endonuclease.