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

Roberto Meidler, Ilan Morad, Michal Amitsur, Hachiro Inokuchi, Gabriel Kaufmann

Research output: Contribution to journalArticlepeer-review

Abstract

The tRNA(Lys)-specific anticodon nuclease exists in latent form in Escheri chia 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 tRNA(Lys) 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 tRNA(Lys) 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 tRNA(Lys). Taken together, the data suggest that the anticodon of tRNA(Lys) harbours anticodon nuclease identity elements and implicates a conserved region in PrrC in their recognition.

Original languageEnglish
Pages (from-to)499-510
Number of pages12
JournalJournal of Molecular Biology
Volume287
Issue number3
DOIs
StatePublished - 2 Apr 1999

Keywords

  • DNA restriction-modification
  • Polynucleotide kinase
  • RNA ligase
  • tRNA identity
  • tRNA splicing endonuclease

Fingerprint

Dive into the research topics of 'Detection of anticodon nuclease residues involved in tRNA(Lys) cleavage specificity'. Together they form a unique fingerprint.

Cite this