Bacterial Toxin RelE: A Highly Efficient Ribonuclease with Exquisite Substrate Specificity Using Atypical Catalytic Residues

Department of Molecular Biophysics and Biochemistry, Yale University , New Haven, Connecticut 06511, United States.
Biochemistry (Impact Factor: 3.02). 11/2013; 52(48). DOI: 10.1021/bi401325c
Source: PubMed


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(2)- and 10(6)-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.

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    • "Because of the tight interaction between a toxin and its cognate antitoxin, high concentrations of denaturant are often necessary [16,28,64–67]. By truncating the antitoxin, the interaction between toxin and antitoxin can be weakened such that the antitoxin can still bind and alleviate toxicity in vivo, but that less denaturing agent is required to dissociate the complex [68]. "
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    ABSTRACT: Toxin-antitoxin (TA) modules are stress response elements that are ubiquitous in the genomes of bacteria and archaea. Production and subsequent purification of individual TA proteins is anything but straightforward as over-expression of the toxin gene is lethal to bacterial and eukaryotic cells and over-production of the antitoxin leads to its proteolytic degradation because of its inherently unstructured nature. Here we describe an effective production and purification strategy centered on an on-column denaturant-induced dissociation of the toxin-antitoxin complex. The success of the method is demonstrated by its application on four different TA families, encoding proteins with distinct activities and folds. A series of biophysical and in vitro activity tests show that the purified proteins are of high quality and suitable for structural studies. Copyright © 2015. Published by Elsevier Inc.
    Protein Expression and Purification 01/2015; 108C:30-40. DOI:10.1016/j.pep.2015.01.001 · 1.70 Impact Factor
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    • "antitoxins (RelB) and one toxin molecule (RelE) bounded together [24] [30]. Therefore, it can be assumed that toxic effect of Xn-relE toxin neutralization may require more than one Xn-relEAT protein molecule. "
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    ABSTRACT: Bacterial toxin-antitoxin (TA) complexes induce programmed cell death and also function to relieve cell from stress by various response mechanisms. Escherichia coli RelB-RelE TA complex consists of a RelE toxin functionally counteracted by RelB antitoxin. In the present study, a novel homolog of RelE toxin designated as Xn-relE toxin from Xenorhabdus nematophila possessing its own antitoxin designated as Xn-relEAT has been identified. Expression and purification of recombinant proteins under native conditions with GST and Ni-NTA chromatography prove the existence of novel TA module. The expression of recombinant Xn-relE under tightly regulated ara promoter in E. coli Top 10 cells confirms its toxic nature in endogenous toxicity assay. The neutralization activity in endogenous toxicity assay by Xn-relEAT antitoxin confirms its antidote nature when studying the whole TA operon under ara regulated promoter. This study promotes newly discovered TA module to be regarded as important as other proteins of type II toxin-antitoxin system.
    The Scientific World Journal 11/2014; 2014:428159. DOI:10.1155/2014/428159 · 1.73 Impact Factor
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    ABSTRACT: Bacteria encounter environmental stresses that regulate a gene expression program required for adaptation and survival. Here, we report the 1.8-Å crystal structure of the Escherichia coli toxin-antitoxin complex YafQ-(DinJ)2-YafQ, a key component of the stress response. The antitoxin DinJ dimer adopts a ribbon-helix-helix motif required for transcriptional autorepression, and toxin YafQ contains a microbial RNase fold whose proposed active site is concealed by DinJ binding. Contrary to previous reports, our studies indicate that equivalent levels of transcriptional repression occur by direct interaction of either YafQ-(DinJ)2-YafQ or a DinJ dimer at a single inverted repeat of its recognition sequence that overlaps with the −10 promoter region. Surprisingly, multiple YafQ-(DinJ)2-YafQ complexes binding to the operator region do not appear to amplify the extent of repression. Our results suggest an alternative model for transcriptional autorepression that may be novel to DinJ-YafQ.
    Journal of Biological Chemistry 06/2014; 289(30). DOI:10.1074/jbc.M114.573006 · 4.57 Impact Factor
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