Biochemical analysis of Thermotoga maritima ribonuclease III and its ribosomal RNA substrates

The site-specific; cleavage of double-stranded (ds) RNA is a conserved early step in bacterial ribosomal RNA (rRNA) maturation that is carried out by ribonuclease III. Studies on the RNase III mechanism of dsRNA cleavage have focused mainly on the enzymes from mesophiles such as Escherichia coli. In contrast, little is known of the RNA processing pathways and the functions of associated ribonucleases in the hyperthermophiles. Therefore, structural and biochemical studies of proteins from hyperthermophilic bacteria are providing essential insight on the sources of biomolecular thermostability, and how enzymes function at high temperatures.;The biochemical behavior of RNase III of the hyperthermophilic bacterium Thermotoga maritima is analyzed using purified recombinant enzyme and the cognate pre-ribosomal; RNAs as substrates. The T. maritima genome encodes a ∼5,000 nucleotide (nt) transcript, expressed from the single ribosomal RNA (rRNA) operon. RNase III processing sites are expected to form through base-pairing of complementary sequences that flank the 16S and 23S rRNAs. The Thermotoga pre--16S and pre--23S processing stems are synthesized in the form of small hairpins, and are efficiently and site-specifically cleaved by Tm-RNase III at sites consistent with an in vivo role of the enzyme in producing the immediate precursors to the mature rRNAs. T. maritima (Tm)-RNase III activity is dependent upon divalent metal ion, with Mg2+ as the preferred species, at concentrations ≥ 1 mM. Mn2+, Co2+ and Ni2+ also support activity, but with reduced efficiency. The enzyme activity is also supported by salt (Na+, K +, or NH4+) in the 50--80 mM range, with an optimal pH of ∼8. Catalytic activity exhibits a broad temperature maximum of ∼40--70°C, with significant activity retained at 95°C. Comparison of the Charged-versus-Polar (CvP) bias of the protein side chains indicates that Tm-RNase III thermostability is due to large CvP bias.;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 determinant of recognition of E. coli (Ec)-RNase III substrates. The pb sequence dependence of reactivity is similar to that observed with the Ec-RNase III pb. Moreover, Tm-RNase III cleaves an Ec-RNase III substrate with identical specificity, and is inhibited by pb antideterminants that also inhibit Ec-RNase III. These studies reveal the conservation acrosss a broad phylogenetic distance of substrate reactivity epitopes, both the positive and negative determinants, among bacterial RNase III substrates.