ABSTRACT
The bacterial tRNA(Lys)-specific PrrC-anticodon nuclease cleaves its natural substrate 5' to the wobble base, yielding 2',3'-cyclic phosphate termini. Previous work has implicated the anticodon of tRNA(Lys) as a specificity element and a cluster of amino acid residues at the carboxy-proximal half of PrrC in its recognition. We further examined these assumptions by assaying unmodified and hypomodified derivatives of tRNA(Lys) as substrates of wild-type and mutant alleles of PrrC. The data show, first, that the anticodon sequence and wobble base modifications of tRNA(Lys) play major roles in the interaction with anticodon nuclease. Secondly, a specific contact between the substrate recognition site of PrrC and the tRNA(Lys) wobble base is revealed by PrrC missense mutations that suppress the inhibitory effects of wobble base modification mutations. Thirdly, the data distinguish between the anticodon recognition mechanisms of PrrC and lysyl-tRNA synthetase.
Subject(s)
Anticodon/genetics , Bacterial Proteins/metabolism , Escherichia coli Proteins , Escherichia coli/enzymology , RNA, Transfer, Lys/genetics , RNA, Transfer, Lys/metabolism , Ribonucleases/metabolism , Alleles , Amino Acid Sequence , Anticodon/metabolism , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Base Sequence , Binding Sites , Escherichia coli/genetics , Escherichia coli/growth & development , Genes, Bacterial/genetics , Genes, Bacterial/physiology , Lysine-tRNA Ligase/metabolism , Molecular Sequence Data , Mutation, Missense/genetics , Point Mutation/genetics , Protein Structure, Secondary , RNA, Bacterial/genetics , RNA, Bacterial/metabolism , Ribonucleases/chemistry , Ribonucleases/genetics , Substrate Specificity , Suppression, Genetic/genetics , tRNA Methyltransferases/genetics , tRNA Methyltransferases/metabolismABSTRACT
The tRNALys-specific anticodon nuclease exists in latent form in Escherichia coli strains containing the optional prr locus. The latency is a result of a masking interaction between the anticodon nuclease core-polypeptide PrrC and the Type IC DNA restriction-modification enzyme EcoprrI. Activation of the latent enzyme by phage T4-infection elicits cleavage of tRNALys 5' to the wobble base, yielding 5'-OH and 2', 3'-cyclic phosphate termini. The N-proximal half of PrrC has been implicated with (A/G) TPase and EcoprrI interfacing activities. Therefore, residues involved in recognition and cleavage of tRNALys were searched for at the C-half. Random mutagenesis of the low-G+C portion encoding PrrC residues 200-313 was performed, followed by selection for loss of anticodon nuclease-dependent lethality and production of full-sized PrrC-like protein. This process yielded a cluster of missense mutations mapping to a region highly conserved between PrrC and two putative Neisseria meningitidis MC58 homologues. This cluster included two adjacent members that relaxed the inherent enzyme's cleavage specificity. We also describe another mode of relaxed specificity, due to mere overexpression of PrrC. This mode was shared by wild-type PrrC and the other mutant alleles. The additional substrates recognised under the promiscuous conditions had, in general, anticodons resembling that of tRNALys. Taken together, the data suggest that the anticodon of tRNALys harbours anticodon nuclease identity elements and implicates a conserved region in PrrC in their recognition.
