Structural alterations in mutant precursors of the yeast tRNALeu3 gene which behave as defective substrates for a highly purified splicing endoribonuclease.

Abstract

We have produced a highly purified preparation of the Xenopus laevis splicing endonuclease (XlaI RNase). The purified enzyme correctly cleaves tRNA precursors, creating substrates for subsequent ligation. The 5'-half molecules have a 2',3' cyclic phosphate at their 3' termini. Assuming that splicing enzymes recognize primarily structural elements in the 'mature domain', we have been studying the conformation of three splicing-defective precursors made from mutants of the yeast tRNALeu3 gene. The mutations alter base-pairing in the D-stem region and two of the mutants are absolute defectives. Enzymatic probing of the structures of the altered tRNA precursors shows that the structural perturbations in these mutants are localized on the 'inside' of the 'L'-shaped three-dimensional structure. The implications of this finding for the recognition process are discussed.