First structural investigation of the restriction ribonuclease RegB: NMR spectroscopic conditions, 13C/15N double-isotopic labelling and two-dimensional heteronuclear spectra.
The bacteriophage T4 genome-encoded ribonuclease RegB is the unique well-defined restriction endoribonuclease. This protein cleaves with an almost absolute specificity its RNA substrate in the middle of the GGAG tetranucleotide mainly found in the Shine-Dalgarno sequence (required for the prokaryotic initiation of the translation). This protein has no significant homology to any known ribonuclease and its structure has never been investigated. The extreme toxicity of this ribonuclease prevents the expression of large quantities for structural studies. Here, we show that the toxicity of RegB can be bypassed by using the RegB H48A point mutant and explain why resolving the structure of this mutant is relevant. For nuclear magnetic resonance (NMR) purposes, we report the preparation of highly pure (13)C/(15)N double-labelled 1.2mM samples of RegB H48A using a high yield expression procedure in minimal medium (30 mg/L). We also present a set of solution conditions that maintain the concentrated samples of this protein stable for long periods at the NMR-required temperature. Finally, we present the first (1)H/(15)N and (1)H/(13)C two-dimensional NMR spectra of RegB H48A. These spectra show that the protein is folded and that the full structural analysis of RegB by NMR is feasible.
Saïda
Fakhri
F
Laboratoire ICSN-RMN, Institut de Chimie des Substances Naturelles, Ecole polytechnique, route de Saclay, 91128 Palaiseau Cedex, France. fsaida@biomail.ucsd.edu
Odaert
Benoît
B
Uzan
Marc
M
Bontems
François
F
D019943:Amino Acid Substitution; D002021:Buffers; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D004722:Endoribonucleases / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007553:Isotope Labeling / Q000379:methods*; D016297:Mutagenesis, Site-Directed; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D009994:Osmolar Concentration; D017354:Point Mutation / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae / Q000235:genetics; D013696:Temperature; D014170:Transformation, Genetic / Q000235:genetics; D014316:Tritium
D002021:Buffers; D009587:Nitrogen Isotopes; D011994:Recombinant Proteins; D014316:Tritium; D004722:Endoribonucleases; C407103:T4 RegB endoribonuclease