IDENTIFICATION OF 3 CORE REGIONS ESSENTIAL FOR PROTEIN SPLICING OF THE YEAST VMA1 PROTOZYME - A RANDOM MUTAGENESIS STUDY OF THE ENTIRE VMA1-DERIVED ENDONUCLEASE SEQUENCE

The translation product of the VMA1 gene of Saccharomyces cerevisiae u ndergoes protein splicing, in which the intervening region is autocata lytically excised and the franking regions are ligated. The splicing r eaction is catalyzed essentially by the in-frame insert, VMA1-derived endonuclease (VDE), which is a site-specific endonuclease to mediate g ene homing, Previous mutational analysis of the splicing reaction has been concentrated extensively upon the splice junctions, However, it s till remains unknown which amino acid residues are crucial for the spl icing reaction within the entire region of VDE and ifs neighboring ele ments. Tn this work, a polymerase chain reaction-based random mutagene sis strategy mas used to identify such residues throughout the overall intervening sequence of the VMA1 gene, Splicing-defective mutant prot eins were initially screened using a bacterial expression system and t hen analyzed further in yeast cells, Mutations were mapped at the N- a nd C-terminal splice junctions and around the N-terminal one-third of VDE, We identified four potent mutants that yielded aberrant products with molecular masses of 200, 90, and 80 kDa. We suggest that the cons erved His(362), newly identified as the essential residue for the spli cing reaction, contributes to the first cleavage at. the N-terminal ju nction, whereas His(736) assists the second cleavage by Asn cyclizatio n at the C-terminal junction, Mutations in these regions did not appea r to destroy the endonuclease activity of VDE.