Ribonuclease III cleavage of a bacteriophage T7 processing signal. Divalent cation specificity, and specific anion effects

Escherlchla coll ribonuclease III, purified to homogeneity from an overexpresslng bacterial strain, exhibits a high catalytic efficiency and thermostable processing activity in vitro. The RNase Ill-catalyzed cleavage of a 47 nucleotide substrate (R1.1 RNA), based on the bacteriophage T7 R1.1 processing signal, follows substrate saturation kinetics, with a K_m of 0.26 μM, and k_cat, of 7.7 mln.^-1 (37°C, In buffer containing 250 mM potassium glutamate and10 mM MgCl₂. Mn²⁺ and Co²⁺ can support the enzymatic cleavage of the R1.1 RNA canonical site, and both metal Ions exhibit concentration dependences similar to that of Mg²⁺ . Mn²⁺ and Co²⁺ in addition promote enzymatic cleavage of a secondary site In R1.1 RNA, which is proposed to result from the altered hydrolytic activity of the metalloenzyme (RNase III 'star' activity), exhibiting a broadened cleavage specificity. Neither Ca²⁺ nor Zn²⁺ support RNase III processing, and Zn²⁺ moreover Inhibits the Mg²⁺ -dependent enzymatic reaction without blocking substrate binding. RNase III does not require monovalent salt for processing activity; however, the in vitro reactivity pattern is influenced by the monovalent salt concentration, as well as type of anion. First, R1.1 RNA secondary site cleavage Increases as the salt concentration is lowered, perhaps reflecting enhanced enzyme binding to substrate. Second, the substitution of glutamate anion for chloride anion extends the salt concentration range within which efficient processing occurs. Third, fluoride anion inhibits RNase III-catalyzed cleavage, by a mechanism which does not Involve inhibition of substrate binding.