The phage T4 restriction endoribonuclease RegB: a cyclizing enzyme that requires two histidines to be fully active

Abstract

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.

Figure 1

Proposed acid–base catalysis of phosphodiester bond cleavage by barnase, RNase A, RNase T1 and RNase T2. HB-Enz. and ^–B-Enz. (Enz-HA and Enz-A^–) denote, respectively, the acidic and the basic forms of the residue acting as a general base catalyst (general acid catalyst).

Figure 2

Detection, by ³¹P NMR, of a 2′,3′-cyclic phosphorus product during the cleavage of the substrate Selex22tb RNA by RegB. (A) Cleavage by the wild-type RegB protein. (B) Negative control experiment using the inactive point mutant RegB R52L. The asterisk indicates the position of the hydrolyzed form of the cyclic product. A peak at this position could be observed provided that the experiment was recorded for a much longer time.

Figure 3

Sequence alignment of the RegB proteins from phage T4 and phage RB69. Four histidines (H42, H48, H68 and H73) out of five are conserved.

Figure 4

In vivo toxicity test of RegB mutants. The viability of E.coli strain BL21(DE3) was checked after transformation with plasmids pARNU2, pERL52, pH42A, pH48A, pH68A and pH73A expressing, respectively, the wild-type RegB protein, the inactive point mutant R52L (positive control) and the four histidine-to-alanine mutants. The only mutants that are maintained in BL21(DE3) are H48A and H68A.

Figure 5

Cleavage reaction kinetics of the Selex22tb RNA by different forms of RegB. (A) The 21 nt Selex22tb RNA is cleaved specificially by RegB in the middle of the GGAG motif, leaving 15 and 6 nt fragments. These fragments are separated on a 20% polyacrylamide gel and stained with ethidium bromide. The light emission of the UV-excited ethidium bromide, in RNA, was recorded using a Vilbert Lourmat Imager and quantified using Bio1D V99 software. (B) Example of two UV-excited gels showing the cleavage of the Selex22tb RNA by the wild-type and the H68A RegB forms. (C) Appearance of the 15 nt product during cleavage of Selex22tb RNA by different forms of RegB (wild type, H42A, H48A, R52L, H68A and H73A).

Figure 6

¹H/¹⁵N HSQC spectra of the R52L, H48A and H68A RegB mutants recorded at 25°C on a Bruker DRX600 spectrometer. The encircled peaks in the spectrum of RegB H48A are found in the same positions in the spectra of the two independent mutants RegB R52L and RegB H68A.