SITE-DIRECTED MUTAGENESIS OF THE BASE RECOGNITION LOOP OF RIBONUCLEASE FROM BACILLUS-INTERMEDIUS (BINASE)

Members of the microbial guanyl-specific ribonuclease family show a hi gh level of structural homology, The structural basis for guanyl base binding by microbial ribonucleases has been established for all member s of the family and the existence of a guanine recognition loop was sh own, However, bacillar RNases such as binase and barnase show far less specificity towards the guanyl base in hydrolysing oligonucleotides c omposed of more than 4 or 5 nucleotides. Using site-directed mutagenes is we introduced a number of amino acid substitutions into the base re cognition loop of binase, The donor sequence originated from the guany l specific ribonuclease Sa, Two single, two double and one triple (ent ire loop substitution) mutants were constructed and overproduced in E. coli, The kinetic properties of the mutant variants are different fro m the wild-type protein, Amino acid substitutions R61V, G60S, S56Q/R61 V, G60S/R61V show 3-fold, 7-fold, 4-fold and 12-fold increased guanyl specificity respectively, However, all mutants retain the ability to c atalyse the hydrolysis of a poly(A) substrate.