The phage T4 restriction endoribonuclease RegB: a cyclizing enzyme that requires two histidines to be fully active.
The regB gene, from the bacteriophage T4, codes for an endoribonuclease that controls the expression of a number of phage early genes. The RegB protein cleaves its mRNA substrates with an almost absolute specificity in the middle of the tertranucleotide GGAG, making it a unique well-defined restriction endoribonuclease. This striking protein has no homology to any known RNase and its catalytic mechanism has never been investigated. Here, we show, using 31P nuclear magnetic resonance (NMR), that RegB produces a cyclic 2',3'-phosphodiester product. In order to determine the residues crucial for its activity, we prepared all the histidine-to- alanine point mutants of RegB. The activity of these mutants was characterized both in vivo and in vitro. In addition, their binding capability was quantified by surface plasmon resonance and their structural integrity was probed by 1H/15N NMR correlation spectroscopy. The results obtained show that only the H48A and the H68A substitutions significantly reduce RegB activity without changing its ability to bind the substrate or affecting its overall structure. Altogether, our results define RegB as a new cyclizing RNase and present His48 and His68 as potent catalytic residues. The effect of the in vivo selected R52L mutation is also described and discussed.
Saïda
Fakhri
F
Laboratoire ICSN-RMN, Institut de Chimie des Substances Naturelles, Ecole polytechnique, Route de Saclay, 91128 Palaiseau, France.
Uzan
Marc
M
Bontems
François
F
D000409:Alanine / Q000235:genetics; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D020134:Catalytic Domain; D003500:Cyclization; D004722:Endoribonucleases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation; D006639:Histidine / Q000235:genetics / Q000502:physiology*; D007700:Kinetics; D008956:Models, Chemical; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D009843:Oligoribonucleotides / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D016415:Sequence Alignment
D009843:Oligoribonucleotides; D006639:Histidine; D004722:Endoribonucleases; C407103:T4 RegB endoribonuclease; D000409:Alanine