Mechanism of Action of Escherichia coli Ribonuclease III. Stringent Chemical Requirement for the Glutamic Acid 117 Side Chain and Mn²⁺ Rescue of the Glu117Asp Mutant

Abstract

Escherichia coli ribonuclease III (EC 3.1.24) is a double-strand- (ds-) specific endoribonuclease involved in the maturation and decay of cellular, phage, and plasmid RNAs. RNase III is a homodimer and requires Mg(2+) to hydrolyze phosphodiesters. The RNase III polypeptide contains an N-terminal catalytic (nuclease) domain which exhibits eight highly conserved acidic residues, at least one of which (Glu117) is important for phosphodiester hydrolysis but not for substrate binding [Li and Nicholson (1996) EMBO J. 15, 1421-1433]. To determine the side chain requirements for activity, Glu117 was changed to glutamine or aspartic acid. The mutant proteins were purified as (His)(6)-tagged species, and both exhibited normal homodimeric behavior as shown by chemical cross-linking. The Glu117Gln mutant is unable to cleave substrate in vitro under all tested conditions but can bind substrate. The Glu117Asp mutant also is defective in cleavage while able to bind substrate. However, low level activity is observed at extended reaction times and high enzyme concentrations, with an estimated catalytic efficiency approximately 15 000-fold lower than that of RNase III. The activity of the Glu117Asp mutant but not that of the Glu117Gln mutant can be greatly enhanced by substituting Mn(2+) for Mg(2+), with the catalytic efficiency of the Glu117Asp-Mn(2+) holoenzyme approximately 400-fold lower than that of the RNase III-Mn(2+) holoenzyme. For RNase III, a Mn(2+) concentration of 1 mM provides optimal activity, while concentrations >5 mM are inhibitory. In contrast, the Glu117Asp mutant is not inhibited by high concentrations of Mn(2+). Finally, high concentrations of Mg(2+) do not inhibit RNase III nor relieve the Mn(2+)-dependent inhibition. In summary, these experiments establish the stringent functional requirement for a precisely positioned carboxylic acid group at position 117 and reveal two classes of divalent metal ion binding sites on RNase III. One site binds either Mg(2+) or Mn(2+) and supports catalysis, while the other site is specific for Mn(2+) and confers inhibition. Glu117 is important for the function of both sites. The implications of these findings on the RNase III catalytic mechanism are discussed.

RNA Editing in Trypanosoma brucei Requires Three Different Editosomes

“…Mutation of the equivalent residue in Escherichia coli RNase III reduced activity in vitro and increased the K m (3,59,65). Analysis of the crystal structure of A. aeolicus suggests that its corresponding residue is involved in RNase III subunit dimerization (17).…”

YmdB: a stress-responsive ribonuclease-binding regulator of E. coli RNase III activity

“…Interestingly, the RNase III E117 mutation identified by Sun and Nicholson (2001) was also found to abrogate endoribonuclease activity in vitro when tested using Mn . Position 117, which Sun and Nicholson (2001) included in a region they designated as the "inhibitory" site of the enzyme, is also within the RNase III segment we showed to be sufficient for binding to YmdB. Collectively, these findings raise the possibility that Mn 2+ binding at or near position 117 enables YmdB interaction with RNase III, and consequent inhibition of enzymatic activity.…”

E. coli RNase III(E38A) generates discrete-sized products from long dsRNA

“…It was our initial observation that the discreet 23-bp dsRNA product started accumulating in low enzyme to substrate ratios, suggesting that E38A (and by extension WT RNase III) was binding cooperatively to the dsRNA substrate. To test this we designed an experiment that utilized the activity of another mutant, E117D, which binds dsRNA (data not shown) or R1.1 (Sun and Nicholson 2001) tightly but does not cleave, thereby acting to protect the bound RNA. Both of the E117D and E38A substitutions are part of the internal portion of the enzyme and therefore would not be expected to contribute to any protein/protein interactions or any cooperativity.…”

“…To test this idea, we used both E38A and E117D in a digestion reaction with a 900-bp dsRNA as substrate. The RNase III E117D mutant binds tightly to both R1.1 (Sun and Nicholson 2001) and to dsRNA (data not shown) but does not cleave. We conjecture for this experiment that E38A and E117D have the same cooperativity properties (i.e., they either bind to the dsRNA substrate cooperatively or they do not).…”

“…A cluster of six acidic residues-E38, E41, D45, D114, and E117 (for Ec-RNase III) from one subunit and E65 from the partner subunit-is located at either end of the valley (Blaszczyk et al 2001). E38 is involved in protein dimerization; E65 is involved in substrate recognition and scissile-bond selection; D45, D114, and D117 chelate the Mn 2+ ion (Blaszczyk et al 2001;Sun and Nicholson 2001); and E41, D45, D114, and E117 carry out the hydrolysis of the scissile bond (Gan et al 2006).…”