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

Hong lin Li, Bhadrani S. Chelladurai, Kejing Zhang, Allen W. Nicholson

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Abstract

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 Km of 0.26 μM, and kcat, 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.

Original languageEnglish (US)
Pages (from-to)1919-1925
Number of pages7
JournalNucleic Acids Research
Volume21
Issue number8
DOIs
Publication statusPublished - Apr 25 1993
Externally publishedYes

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ASJC Scopus subject areas

  • Genetics

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