A NOVEL STRATEGY FOR STABILIZATION OF ESCHERICHIA-COLI RIBONUCLEASE HI INVOLVING A SCREEN FOR AN INTRAGENIC SUPPRESSOR OF CARBOXYL-TERMINALDELETIONS

A strategy to genetically select Escherichia coli ribonuclease HI muta nts with enhanced thermostability is described. E. coil strain MIC3001 , which shows an RNase H-dependent, temperature-sensitive growth pheno type, was used for this purpose. Introduction of the rnhA gene permits the growth of this temperature-sensitive strain, whereas the gene for the truncated protein, 142-RNase HI, which lacks the carboxyl-termina l 13 residues, cannot. Analyses of the production levels and the stabi lity of a series of mutant proteins with COOH-terminal truncations sug gested that 142-RNase HI is nonfunctional in vivo because of a dramati c decrease in the protein stability. Polymerase chain reaction-mediate d random mutagenesis of the rnhA142 gene, encoding 142-RNase HI, follo wed by selection of revertants, allowed us to isolate 11 single amino acid substitutions that render 142-RNase HI functional in vivo. Of the m, eight substitutions were shown to enhance the thermal stability of the wild-type RNase HI protein, and of these, six were novel. The gene tic selection strategy employed in this experiment was thus shown to b e effective for identifying amino acid substitutions that enhance the thermal stability of E. coil RNase HI. Such a strategy would be versat ile if a protein of interest could be destabilized by a deletion or a truncation and a conditional-lethal strain were available.