A mutagenic analysis of the RNase mechanism of the bacterial Kid toxin by mass spectrometry.
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'-UA(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 the catalytic site abolished RNA cleavage without substantially altering RNA binding. Mutations in residues proposed to be involved in RNA binding show reduced binding efficiency and a corresponding decrease in RNA cleavage efficiency. The cleavage profiles of the different mutants were similar with the two substrates used, but RNA cleavage required much lower protein concentrations when the 5'-UUACU-3' substrate was used. Protein synthesis and growth assays are consistent with there being a correlation between the RNase activity of Kid and its inhibitory potential. These results give important support to the available models of Kid RNase and the Kid-RNA complex.
Diago-Navarro
Elizabeth
E
Centro de Investigaciones Biológicas, Departamento de Microbiología Molecular, Madrid, Spain.
Kamphuis
Monique B
MB
Boelens
Rolf
R
Barendregt
Arjan
A
Heck
Albert J
AJ
van den Heuvel
Robert H
RH
Díaz-Orejas
Ramón
R
D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics; D008958:Models, Molecular; D009154:Mutation; D011485:Protein Binding; D012260:Ribonucleases / Q000737:chemistry* / Q000235:genetics; D021241:Spectrometry, Mass, Electrospray Ionization; D013379:Substrate Specificity
D029968:Escherichia coli Proteins; C473072:kid toxin protein, E coli plasmid R1; D012260:Ribonucleases