Figure 1.
CdiA-CT tRNase activity requires a cofactor. (A) In vitro tRNase assays. S100 lysates or isolated tRNA (purified by either GITC-phenol or DEAE-cellulose chromatography) were incubated with CdiA-CT and CdiI-His6 (where indicated) as described in the Materials and Methods. The samples labeled DEAE tRNA+S100 protein contained a reconstituted S100 lysate in which the separated tRNA and protein fractions were recombined. Reactions were run on denaturing polyacrylamide gels, and tRNAs were visualized by ethidium bromide staining. The migration positions of full-length and cleaved tRNAs are indicated. (B) Purification scheme for enrichment of the CdiA-CT-activating factor. Proteins from E. coli S100 lysates were fractionated by anion exchange (DEAE-cellulose) and hydrophobic interaction (butyl-Sepharose) chromatography as illustrated in the flow chart. The final peak activating fraction was analyzed by SDS-PAGE, and proteins were visualized by staining with Coomassie blue.