Biochemical characterization of YloC ribonuclease activity. (A) Metal-ion dependence of YloC activity. An amount of 30 pmol of RNA oligo (IR800-labeled + unlabeled) was incubated with 10 pmol of YloC for 20 min at 37°C. Abbreviations of divalent cations: (Ca) Calcium, (Cu) copper, (Co) cobalt, (Mg) magnesium, (Mn) manganese, (Zn) zinc. All reactions were with 2.5-mM divalent cation. (C) Control lane with no protein. (B) Initial rate of YloC cleavage, as measured by appearance of 5- to 7-nt product, in the presence of indicated divalent metal cation (2.5 mM) at 37°C. Numbers in parentheses are the initial rate of product formation, relative to the initial rate in the presence of Mg2+ (average of two experiments). (C) Determination of the nature of the 3′ end after YloC cleavage. (Lane 1) IR800-labeled 36-nt RNA oligo. (Lane 2) Thirty-six-nucleotide RNA ligated to 18-nt DNA oligo. (Lane 3) Five-nucleotide to 7-nt products from 36-nt RNA oligo after incubation with YloC for 20 min at 37°C. (Lanes 4,5) Ligation of 5- to 7-nt YloC cleavage products to 18-nt DNA oligo without or with prior polynucleotide kinase (PNK) treatment, as indicated. Sizes of RNA and RNA–DNA products (nucleotides) are indicated at the left.
