TAILIEUCHUNG - Báo cáo khoa học: A mutagenic analysis of the RNase mechanism of the bacterial Kid toxin by mass spectrometry

Kid, the toxin of theparD(kis, kid) maintenance system of plasmid R1, is an endoribonuclease that preferentially cleaves RNA at the 5¢ of A in the core sequence 5¢-UA(A⁄C)-3¢. A model of the Kid toxin interacting with the uncleavable mimetic 5¢-AdUACA-3¢ is available. | A mutagenic analysis of the RNase mechanism of the bacterial Kid toxin by mass spectrometry Elizabeth Diago-Navarro1 Monique B. Kamphuis2 Rolf Boelens2 Arjan Barendregt3 Albert J. Heck3 Robert H. van den Heuvel3 and Ramon Diaz-Orejas1 1 Centro de Investigaciones Biologicas Departamento de Microbiologia Molecular Madrid Spain 2 Bijvoet Center for Biomolecular Research Department of NMR Spectroscopy Utrecht University The Netherlands 3 Bijvoet Center for Biomolecular Research Biomolecular Mass Spectrometry and Proteomics group Utrecht Institute for Pharmaceutical Sciences Utrecht University The Netherlands Keywords Kid mutants Kid RNase model native mass spectrometry protein-RNA binding protein-RNA cleavage Correspondence R. Diaz-Orejas Centro de Investigaciones Biologicas Departamento de Microbiologia Molecular Ramiro de Maeztu 9 E-28040 Madrid Spain Fax 34 915 360 432 Tel 34 918 373 112 E-mail ramondiaz@ Present address Schering-Plough Biotech Quality Unit Oss The Netherlands Received 17 May 2009 revised 1 July 2009 accepted 6 July 2009 doi Kid the toxin of the parD kis kid maintenance system of plasmid R1 is an endoribonuclease that preferentially cleaves RNA at the 5 of A in the core sequence 5 -U A A C -3 . A model of the Kid toxin interacting with the uncleavable mimetic 5 -AdUACA-3 is available. To evaluate this model a significant collection of mutants in some of the key residues proposed to be involved in RNA binding T46 A55 T69 and R85 or RNA cleavage R73 D75 and H17 were analysed by mass spectrometry in RNA binding and cleavage assays. A pair of substrates 5 -AUACA-3 and its uncleavable mimetic 5 -AdUACA-3 used to establish the model and structure of the Kid-RNA complex were used in both the RNA cleavage and binding assays. A second RNA substrate 5 -UUACU-3 efficiently cleaved by Kid both in vivo and in vitro was also used in the cleavage assays. Compared with the wild-type protein mutations in the residues of .

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