Detection of anticodon nuclease residues involved in tRNA(Lys) cleavage specificity

The tRNA(Lys)-specific anticodon nuclease exists in latent form in Escheric hia coli strains containing the optional pur locus. The latency is a result of a masking interaction between the anticodon nuclease core-polypeptide P rrC and the Type IC DNA restriction-modification enzyme EcoprrI. Activation of the latent enzyme by phage T4-infection elicits cleavage of tRNA(Lys) 5 ' to the wobble base, yielding 5'-OH and 2',3'-cyclic phosphate termini. Th e N-proximal half of PrrC has been implicated with (A/G) TPase and EcoprrI interfacing activities. Therefore, residues involved in recognition and cle avage of tRNA(Lys) were searched for at the C-half. Random mutagenesis of t he low-G + C portion encoding PrrC residues 200-313 was performed, followed by selection for loss of anticodon nuclease-dependent lethality and produc tion of full-sized PrrC-like protein. This process yielded a cluster of mis sense mutations mapping to a region highly conserved between PrrC and two p utative Neisseria meningitidis MC58 homologues. This cluster included two a djacent members that relaxed the inherent enzyme's cleavage specificity. We also describe another mode of relaxed specificity, due to mere overexpress ion of PrrC. This mode was shared by wild-type PrrC and the other mutant al leles. The additional substrates recognised under the promiscuous condition s had, in general, anticodons resembling that of tRNA(Lys). Taken together, the data suggest that the anticodon of tRNA(Lys) harbours anticodon nuclea se identity elements and implicates a conserved region in PrrC in their rec ognition. (C) 1999 Academic Press.