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Biochemistry. 2014 Jan 14;53(1):188-201. doi: 10.1021/bi4008098. Epub 2013 Dec 23.

Light activation of Staphylococcus aureus toxin YoeBSa1 reveals guanosine-specific endoribonuclease activity.

Author information

1
Department of Chemistry, University of Illinois at Urbana-Champaign , Urbana, Illinois 61801, United States.

Abstract

The Staphylococcus aureus chromosome harbors two homologues of the YefM-YoeB toxin-antitoxin (TA) system. The toxins YoeBSa1 and YoeBSa2 possess ribosome-dependent ribonuclease (RNase) activity in Escherichia coli. This activity is similar to that of the E. coli toxin YoeBEc, an enzyme that, in addition to ribosome-dependent RNase activity, possesses ribosome-independent RNase activity in vitro. To investigate whether YoeBSa1 is also a ribosome-independent RNase, we expressed YoeBSa1 using a novel strategy and characterized its in vitro RNase activity, sequence specificity, and kinetics. Y88 of YoeBSa1 was critical for in vitro activity and cell culture toxicity. This residue was mutated to o-nitrobenzyl tyrosine (ONBY) via unnatural amino acid mutagenesis. YoeBSa1-Y88ONBY could be expressed in the absence of the antitoxin YefMSa1 in E. coli. Photocaged YoeBSa1-Y88ONBY displayed UV light-dependent RNase activity toward free mRNA in vitro. The in vitro ribosome-independent RNase activity of YoeBSa1-Y88ONBY, YoeBSa1-Y88F, and YoeBSa1-Y88TAG was significantly reduced or abolished. In contrast to YoeBEc, which cleaves RNA at both adenosine and guanosine with a preference for adenosine, YoeBSa1 cleaved mRNA specifically at guanosine. Using this information, a fluorometric assay was developed and used to determine the kinetic parameters for ribosome-independent RNA cleavage by YoeBSa1.

PMID:
24279911
PMCID:
PMC3927539
DOI:
10.1021/bi4008098
[Indexed for MEDLINE]
Free PMC Article

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