RNase activity in vitro. (A) Assay for RNase activity in a dialyzed protein extract from BG505, the quadruple 3′ exonuclease mutant strain. [IR800]5′-end-labeled RNA oligonucleotide (oligo) incubated at 37°C for time (in minutes) indicated above each lane, with or without added inorganic phosphate. T1 lane is the same oligo digested with RNase T1, which cleaves after G residues. Numbers at the left indicate migration of T1 digestion products. Migration of full-length 36-nt RNA (FL) and limit digestion products of 5–7 nt indicated at the right. (B) Test of candidate proteins for RNase activity. IR800-labeled RNA oligo was incubated for 30 min at 37°C. (C) Control lane with no added protein. Purified 8×His-Sumo-tagged proteins indicated above each lane. (Vector lane) RNase digest with protein from a strain carrying the pET-57 vector without the insert. (Extract lane) RNase digest with protein extract from BG505.
