Nucleotide substrates of Gld2. Gld2 was incubated with varying nucleotides and the precursor microRNA let-7a or mature microRNA let-7a-5p. (A) Addition of single [α-32P]-labeled nucleotides to pre-let-7a and Gld2 as indicated. (B) Competitive nucleotide addition: In a competition assay, Gld2 was incubated with pre-let-7a in the presence of all four unlabeled nucleotides in equimolar amounts with a portion of the indicated nucleotide in an [α-32P]-labeled form. (C) 5′ end phosphorylation: Gld2 activity on let-7a-5p substrates with differentially phosphorylated 5′ ends were assayed with [α-32P]-ATP. (5′-no p) unphosphorylated, (5′-p) monophosphate, (5′-pp) diphosphate, (5′-ppp) triphosphate.
