Ribonuclease III cleavage of a bacteriophage T7 processing signal. Divalent cation specificity, and specific anion effects.
Escherichia coli ribonuclease III, purified to homogeneity from an overexpressing bacterial strain, exhibits a high catalytic efficiency and thermostable processing activity in vitro. The RNase III-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 Km of 0.26 microM, and kcat of 7.7 min.-1 (37 degrees C, in buffer containing 250 mM potassium glutamate and 10 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 Mg(2+)-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.
Li
H L
HL
Department of Biological Sciences, Wayne State University, Detroit, MI 48202.
Chelladurai
B S
BS
Zhang
K
K
Nicholson
A W
AW
D000838:Anions; D017123:Bacteriophage T7 / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D002412:Cations; D004279:DNA, Viral; D004722:Endoribonucleases / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology; D029968:Escherichia coli Proteins; D007700:Kinetics; D008670:Metals / Q000378:metabolism; D008969:Molecular Sequence Data; D012323:RNA Processing, Post-Transcriptional; D012367:RNA, Viral / Q000378:metabolism*; D043244:Ribonuclease III; D012492:Salts; D013379:Substrate Specificity
D000838:Anions; D002412:Cations; D004279:DNA, Viral; D029968:Escherichia coli Proteins; D008670:Metals; D012367:RNA, Viral; D012492:Salts; D004722:Endoribonucleases; D043244:Ribonuclease III; C468366:ribonuclease III, E coli