Bacterial Toxin RelE: A Highly Efficient Ribonuclease
with Exquisite Substrate Specificity Using Atypical Catalytic Residues
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Abstract
The toxin RelE is a ribosome-dependent
endoribonuclease implicated
in diverse cellular processes, including persistence. During amino
acid starvation, RelE inhibits translation by cleaving ribosomal A-site
mRNA. Although RelE is structurally similar to other microbial endoribonucleases,
the active-site amino acid composition differs substantially and lacks
obvious candidates for general acid–base functionality. Highly
conserved RelE residues (Lys52, Lys54, Arg61, Arg81, and Tyr87) surround
the mRNA scissile phosphate, and specific 16S rRNA contacts further
contribute to substrate positioning. We used a single-turnover kinetic
assay to evaluate the catalytic importance of individual residues
in the RelE active site. Within the context of the ribosome, RelE
rapidly cleaves A-site mRNA at a rate similar to those of traditional
ribonucleases. Single-turnover rate constants decreased between 10<sup>2</sup>- and 10<sup>6</sup>-fold for the RelE active-site mutants
of Lys52, Lys54, Arg61, and Arg81. RelE may principally promote catalysis
via transition-state charge stabilization and leaving-group protonation,
in addition to achieving in-line substrate positioning in cooperation
with the ribosome. This kinetic analysis complements structural information
to provide a foundation for understanding the molecular mechanism
of this atypical endoribonuclease