Molecular dissection of mitogillin reveals that the fungal ribotoxins are a family of natural genetically engineered ribonucleases

Mitogillin and the related fungal ribotoxins are highly specific ribonuclea ses which inactivate the ribosome enzymatically by cleaving the 23-28 S RNA of the large ribosomal subunit at a single phosphodiester bond. The site o f cleavage occurs between G(4325) and A(4326) (rat ribosome numbering) whic h are present in one of the most conserved sequences (the alpha-sarcin loop ) among the large subunit ribosomal RNAs of all living species. Amino acid sequence comparison of ribotoxins and guanyl/purine ribonucleases have iden tified domains or residues likely involved in ribonucleolytic activity or c leavage specificity. Fifteen deletion mutants (each 4 to 8 amino acid delet ions) in motifs of mitogillin showing little amino acid sequence homology w ith guanyl/purine ribonucleases were constructed by site-directed mutagenes is, Analyses of the purified mutant proteins identified those regions in fu ngal ribotoxins contributing to ribosome targeting and modulating the catal ytic activity of the toxin; some of the identified motifs are homologous to sequences in ribosomal proteins and elongation factors. This mutational st udy of mitogillin together with the recently published x-ray structure of r estrictocin (a close relative of mitogillin) supports the hypothesis that t he specific cleavage properties of ribotoxins are the result of natural gen etic engineering in which the ribosomal targeting elements of ribosome-asso ciated proteins were inserted into nonessential regions of T1-like ribonucl eases.