Non-cytotoxic variants of the Kid protein that retain their auto-regulatory activity

Santos-Sierra, Sandra; Lemonnier, Marguerite; Núñez, B; Hargreaves, David; Rafferty, John B.; Giraldo, Rafael; Andreu, José Manuel; Dı́az-Orejas, Ramón

doi:10.1016/s0147-619x(03)00048-9

Cited by 15 publications

(38 citation statements)

References 22 publications

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“…It is therefore functionally similar to Q11 in RNase A and to Y38 in RNase T1. 24 H17 was not observed to be essential for Kid toxicity in the previously reported mutagenesis study, 14,29 but the method used allows only histidine to tyrosine mutations. A H17Y mutant would retain hydrogen-bonding capacity and would therefore not affect Kid cleavage activity significantly.…”

Section: Kid-rna Interactions Responsible For Rna Cleavagementioning

confidence: 79%

“…Previously, a number of non-toxic Kid mutants has been obtained. 14,29 The residues identified in the mutagenesis study to be essential for the toxicity of the Kid protein almost all belong to clusters #1, #4 and #5 of the described RNAbinding pocket of Kid. Only the buried residue T29 does not show a chemical shift perturbation, and residue P94 could not be observed in the NMR spectra because it lacks an amide proton.…”

Section: Resultsmentioning

confidence: 99%

“…The negatively charged residues determined to be essential for Kid toxicity Model for RNA Binding and Catalytic Site of Kid in the previously reported mutagenesis study are E18, D75 and D81, whereas the positively charged residues are R73 and R85. 29 In that study, it was shown that E18K, R73H, D75N, D81N, R85W and R85Q mutations resulted in inactive Kid. On the basis of this information, two additional AIRs were defined (see Materials and Methods for details).…”

Section: A Model Of the Kid-rna Complexmentioning

confidence: 95%

“…14, 29 The large arginine side-chain most likely occupies the space needed for a nucleotide at this position.…”

Section: Kid-rna Interactions Involved In Rna Recognition and Bindingmentioning

confidence: 99%

“…It has been reported that disruption of the salt-bridge between E18 of monomer A and R85 of monomer B would cause a substantial destabilisation of the dimer interface. 14,29 Since this interface is part of the concatenated RNA-binding pocket needed for Kid activity, neither E18 nor R85 is able to participate in Kid cleavage. Residue D81 of monomer B points away from the RNAbinding pocket and forms a hydrogen bond with the backbone of H17 of monomer A.…”

Section: Kid-rna Interactions Responsible For Rna Cleavagementioning

confidence: 99%

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Model for RNA Binding and the Catalytic Site of the RNase Kid of the Bacterial parD Toxin–Antitoxin System

Kamphuis

Bonvin

Monti

et al. 2006

Journal of Molecular Biology

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The toxin Kid and antitoxin Kis are encoded by the parD operon of Escherichia coli plasmid R1. Kid and its chromosomal homologues MazF and ChpBK have been shown to inhibit protein synthesis in cell extracts and to act as ribosome-independent endoribonucleases in vitro. Kid cleaves RNA preferentially at the 5 0 side of the A residue in the nucleotide sequence 5 0 -UA(A/C)-3 0 of single-stranded regions. Here, we show that RNA cleavage by Kid yields two fragments with a 2 0 :3 0 -cyclic phosphate group and a free 5 0 -OH group, respectively. The cleavage mechanism is similar to that of RNases A and T1, involving the uracil 2 0 -OH group. Via NMR titration studies with an uncleavable RNA mimic, we demonstrate that residues of both monomers of the Kid dimer together form a concatenated RNA-binding surface. Docking calculations based on the NMR chemical shifts, the cleavage mechanism and previously reported mutagenesis data provide a detailed picture of the position of the AUACA fragment within the binding pocket. We propose that residues D75, R73 and H17 form the active site of the Kid toxin, where D75 and R73 are the catalytic base and acid, respectively. The RNA sequence specificity is defined by residues T46, S47, A55, F57, T69, V71 and R73. Our data show the importance of these residues for Kid function, and the implications of our results for related toxins, such as MazF, CcdB and RelE, are discussed.

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Section: Kid-rna Interactions Responsible For Rna Cleavagementioning

confidence: 79%

Section: Resultsmentioning

confidence: 99%

Section: A Model Of the Kid-rna Complexmentioning

confidence: 95%

“…14, 29 The large arginine side-chain most likely occupies the space needed for a nucleotide at this position.…”

Section: Kid-rna Interactions Involved In Rna Recognition and Bindingmentioning

confidence: 99%

Section: Kid-rna Interactions Responsible For Rna Cleavagementioning

confidence: 99%

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Model for RNA Binding and the Catalytic Site of the RNase Kid of the Bacterial parD Toxin–Antitoxin System

Kamphuis

Bonvin

Monti

et al. 2006

Journal of Molecular Biology

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Interactions between the toxin Kid of the bacterial parD system and the antitoxins Kis and MazE

et al. 2007

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Insights into the specificity of RNA cleavage by the Escherichia coli MazF toxin

et al. 2004

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The mazEF (chpA) toxin-antitoxin system of Escherichia coli is involved in the cell response to nutritional and antibiotic stresses as well as in bacterial-programmed cell death. Valuable information on the MazF toxin was derived from the determination of the crystal structure of the MazE/MazF complex and from in vivo data, suggesting that MazF promoted ribosome-dependent cleavage of messenger RNA. However, it was concluded from recent in vitro analyses using a MazF-(His6) fusion protein that MazF was an endoribonuclease that cleaved messenger RNA specifically at 5 0 -ACA-3 0 sites situated in singlestranded regions. In contrast, our work reported here shows that native MazF protein cleaves RNA at the 5 0 side of residue A in 5 0 -NAC-3 0 sequences (where N is preferentially U or A). MazFdependent cleavage occurred at target sequences situated either in single-or double-stranded RNA regions. These activities were neutralized by a His6-MazE antitoxin. Although essentially consistent with previous in vivo reports on the substrate specificity of MazF, our results strongly suggest that the endoribonuclease activity of MazF may be modulated by additional factors to cleave messenger and other cellular RNAs.

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Non-cytotoxic variants of the Kid protein that retain their auto-regulatory activity

Cited by 15 publications

References 22 publications

Model for RNA Binding and the Catalytic Site of the RNase Kid of the Bacterial parD Toxin–Antitoxin System

Model for RNA Binding and the Catalytic Site of the RNase Kid of the Bacterial parD Toxin–Antitoxin System

Interactions between the toxin Kid of the bacterial parD system and the antitoxins Kis and MazE

Insights into the specificity of RNA cleavage by the Escherichia coli MazF toxin

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