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.

A Novel Family of Sequence-specific Endoribonucleases Associated with the Clustered Regularly Interspaced Short Palindromic Repeats

“…RNases can be subdivided into two groups depending on the position of the cleavage of the phosphodiester linkage (36). Enzymes of the first class cleave the bond on the 3Ј-side (producing a 5Ј-phosphorylated second product) and include numerous intracellular endo-and exoribonucleases.…”

Structural and Functional Studies of RegB, a New Member of a Family of Sequence-specific Ribonucleases Involved in mRNA Inactivation on the Ribosome

“…We also assumed that RegB catalytic mechanism involves at least one histidine residue. Accordingly, as previously reported, we mutated each RegB histidine residue to alanine and obtained two non-toxic mutants, H48A and H68A (12). Comparison of the HSQC spectra of these two mutants with that of another independently identified inactive mutant (R52L) indicates that these proteins have the same three-dimensional structure.…”

“…In addition, the H86A mutant conserves 20% of the wild-type activity, and the H48A and H68A (but not R52L) were shown to bind RegB substrates with the same affinity as the wild-type protein. All this strongly suggests that the three mutants are good structural models of the wild-type protein (12). We found that the H68A mutant was difficult to overproduce due to its residual activity but obtained a good production yield (ϳ20 mg⅐liter Ϫ1 ) for the H48A mutant (14), which was thus chosen for further analysis.…”

“…In the absence of evidence for additional sequence determinants, we postulated that RegB may recognize a particular RNA structure, and indeed have shown that a particular secondary structure correlates with RNA susceptibility to cleavage (11). We have also shown that RegB acts as a transphosphorylase, producing 3Ј-phosphate oligoribonucleotides (7,12). Finally, RegB activity as measured in vitro is very low: ϳ10 Ϫ3 mol of substrate are cleaved per mole of enzyme per minute, but this activity is enhanced by a factor up to 100 in the presence of the ribosomal subunit 30 S or of the ribosomal protein S1 (13, 11).…”

“…It cannot be overproduced in E. coli for structural purposes, especially when isotopic enrichment is required (14). We overcame this obstacle by isolating a non-toxic point mutant (RegB-H48A) that retains RegB structural integrity and ability to bind substrates (12). Here, we report the three-dimensional structure of RegB and present a map of the interaction sites between the enzyme and two different RNA substrates.…”