Detection of anticodon nuclease residues involved in tRNALys cleavage specificity.
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.
Meidler
R
R
Department of Biochemistry, Tel Aviv University, Ramat Aviv, 69978, Israel.
Morad
I
I
Amitsur
M
M
Inokuchi
H
H
Kaufmann
G
G
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D017124:Conserved Sequence; D017931:DNA Primers / Q000235:genetics; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D029968:Escherichia coli Proteins; D015870:Gene Expression; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D020125:Mutation, Missense; D010641:Phenotype; D012357:RNA, Transfer, Lys / Q000378:metabolism*; D012260:Ribonucleases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D001426:Bacterial Proteins; D017931:DNA Primers; D029968:Escherichia coli Proteins; D012357:RNA, Transfer, Lys; C084965:PrrC protein, E coli; D012260:Ribonucleases; C049345:anticodon nuclease