Localization of the catalytic activity in restrictocin molecule by deletion mutagenesis

Restrictocin, produced by the fungus Aspergillus restrictus, is a highly sp ecific ribonucleolytic toxin which cleaves a single phosphodiester bond bet ween G4325 and A4326 in the 28S rRNA. It is a nonglycosylated, single-chain , basic protein of 149 amino acids. The putative catalytic site of restrict ocin includes Tyr47, His49, Glu95, Arg120 and His136. To map the catalytic activity in the restrictocin molecule, and to study the role of N- and C-te rminus in its activity, we have systematically deleted amino-acid residues from both the termini. Three N-terminal deletions removing 8, 15 and 30 ami no acids, and three C-terminal deletions lacking 4, 6, and 11 amino acids w ere constructed. The deletion mutants were expressed in Escherichia coli, p urified to homogeneity and functionally characterized. Removal of eight N-t erminal or four C-terminal amino acids rendered restrictocin partially inac tive, whereas any further deletions from either end resulted in the complet e inactivation of the toxin. The study demonstrates that intact N- and C-te rmini are required for the optimum functional activity of restrictocin.