Thermotoga maritima ribonuclease III. Characterization of thermostable biochemical behavior and analysis of conserved base-pairs that function as reactivity epitopes for the Thermotoga 23S ribosomal RNA precursor.

摘要

The cleavage of double-stranded(ds) RNA by ribonuclease III is a conserved early step in bacterial rRNA maturation. Studies on the mechanism of dsRNA cleavage by RNase III have focused mainly on the enzymes from mesophiles such as Escherichia coli. In contrast, neither the catalytic properties of extremophile RNases III, nor the structures and reactivities of their cognate substrates has been described. The biochemical behavior of RNase III of the hyperthermophilic Bacterium Thermotoga maritima was analyzed using purified recombinant enzyme. T. maritima(Tm) RNase III catalytic activity exhibits a broad optimal temperature range of ∼40-70°C, with significant activity at 95°C. Tm-RNase III cleavage of substrate is optimally supported by Mg²⁺ at ≥1 mM concentrations. Mn²⁺, Co²⁺ and Ni²⁺ also support activity, but with reduced efficiencies. The enzyme functions optimally at pH 8, and ∼50-80 mM salt concentrations. Small RNA hairpins that incorporate the 16S and 23S pre-rRNA stem sequences are efficiently cleaved by Tm-RNase III at sites that are consistent with production in vivo of the immediate precursors to the mature rRNAs. Analysis of pre-23S substrate variants reveals a dependence of reactivity on the base-pair (bp) sequence in the proximal box (pb), a site of protein contact that functions as a positive recognition determinant for E. coli(Ec) RNase III substrates. The dependence of reactivity on the pb sequence is similar to that observed with Ec-RNase III substrates. In fact, Tm-RNase III cleaves an Ec-RNase III substrate with identical specificity, and is inhibited by antideterminant bp that also inhibit Ec-RNase III. These results indicate the conservation, across a broad phylogenetic distance, of positive and negative determinants of reactivity of bacterial RNase III substrates.