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Volume 272, Number 25, Issue of June 20, 1997 pp. 15668-15674
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

Identification of Three Core Regions Essential for Protein Splicing of the Yeast Vma1 Protozyme
A RANDOM MUTAGENESIS STUDY OF THE ENTIRE VMA1-DERIVED ENDONUCLEASE SEQUENCE

(Received for publication, October 7, 1996, and in revised form, March 22, 1997)

Masato Kawasaki , Satoru Nogami , Yoshinori Satow , Yoshikazu Ohya and Yasuhiro Anraku

From the Department of Biological Sciences, Graduate School of Science and the  Faculty of Pharmaceutical Sciences, University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113, Japan

The translation product of the VMA1 gene of Saccharomyces cerevisiae undergoes protein splicing, in which the intervening region is autocatalytically excised and the franking regions are ligated. The splicing reaction is catalyzed essentially by the in-frame insert, VMA1-derived endonuclease (VDE), which is a site-specific endonuclease to mediate gene homing. Previous mutational analysis of the splicing reaction has been concentrated extensively upon the splice junctions. However, it still remains unknown which amino acid residues are crucial for the splicing reaction within the entire region of VDE and its neighboring elements. In this work, a polymerase chain reaction-based random mutagenesis strategy was used to identify such residues throughout the overall intervening sequence of the VMA1 gene. Splicing-defective mutant proteins were initially screened using a bacterial expression system and then analyzed further in yeast cells. Mutations were mapped at the N- and 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 conserved His³⁶², newly identified as the essential residue for the splicing reaction, contributes to the first cleavage at the N-terminal junction, whereas His⁷³⁶ assists the second cleavage by Asn cyclization at the C-terminal junction. Mutations in these regions did not appear to destroy the endonuclease activity of VDE.

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