RNA CLEAVAGE WITHOUT HYDROLYSIS - SPLITTING THE CATALYTIC ACTIVITIES OF BINASE WITH ASN101 AND THR101 MUTATIONS

Members of the microbial guanyl-specific ribonuclease family catalyse the endonucleolytic cleavage of single-stranded RNA in a two-step reac tion involving transesterification to form a 2',3'-cyclic phosphate an d its subsequent hydrolysis to yield the respective 3'-phosphate, The extracellular ribonuclease from Bacillus intermedius (binase, RNase Ri ) shares a common mechanism for RNA hydrolysis with mammalian RNases, Two catalytic residues in the active site of binase, Glu72 and His101, are thought to be involved in general acid-general base catalysis of RNA cleavage, Using site-directed mutagenesis, binase mutants were pro duced containing amino acid substitutions H101N and H101T and their ca talytic properties towards RNA, poly(I), poly(A), GPC and guanosine 2' ,3'-cyclic phosphate (cGMP) substrates were studied, The engineered mu tant proteins are active in the transesterification step which produce s the 2',3'-cyclic phosphate species but they have lost the ability to catalyse hydrolysis of the cyclic phosphate to give the 3' monophosph ate product.