LEAD-CATALYZED CLEAVAGE OF YEAST TRANSFER RNAPHE MUTANTS

Yeast tRNAPhe lacking modified nucleotides undergoes lead-catalyzed cleavage between nucleotides U17 and Gl8 at a rate very similar to that of its fully modified counterpart. The rates of cleavage for 28 tRNAPhe mutants were determined to define the structural requirements of this reaction. The cleavage rate was found to be very dependent on the identity and correct positioning of the two lead-coordinating pyrimidines defined by X-ray crystallography. Nucleotide changes that disrupted the tertiary interactions of tRNAphe reduced the rate of cleavage even when they were distant from the lead binding pocket. However, nucleotide changes designed to maintain tertiary interactions showed normal rates of cleavage, thereby making the reaction a useful probe for tRNAPhe structure. Certain mutants resulted in the enhancement of cleavage at a “cryptic” site at C48. The sequences of Escherichia coli tRNAphe and yeast tRNAArg were altered such that they acquired the ability to cleave at U17, confirming our understanding of the structural requirements for cleavage. This mutagenic analysis of the lead cleavage domain provides a useful guide for similar analysis of autocatalytic self-cleavage reactions. © 1990, American Chemical Society. All rights reserved.