FIGURE 3.
ORF240 binding of tRNAThr(CGU) requires G35 and U36. (A) Trm140 specifically binds tRNAThr substrates. Trm140 was purified from a WT strain expressing TRM140-PT (or a vector control) by pull-down using IgG Sepharose, followed by a wash step and by elution of bound Trm140 by 3C protease treatment. Then, copurifying RNAs were resolved by PAGE, transferred to nitrocellulose, and analyzed by hybridization with probes as indicated. V, vector control; T, Trm140-PT. (B) ORF240 binds tRNASer substrates as efficiently as Trm140. Trm140 and ORF240 were purified from WT strains expressing TRM140-PT or ORF240-PT (or a vector control) by pull-down, and copurifying tRNAs were analyzed as in A. O, ORF240-PT. (C) ORF240 binds specifically and reproducibly to tRNAThr(CGU) and not to four nonsubstrate tRNAs. ORF240-PT was expressed in WT cells in biological triplicate and purified by pull-down, two wash steps, and 3C protease elution of bound ORF240, and copurifying tRNAs were analyzed as in A. The percentage of tRNA retained in the combined second wash and the elution step, relative to that in the crude extract was calculated for each tRNA, as indicated. CE, crude extract (0.4% loaded); W1, wash 1 (10% loaded); W2, wash 2, (10% loaded); E, elution (10% loaded). (D) G35 and U36 of tRNAThr(CGU) are important for binding of tRNA to ORF240. Strains containing integrated tRNAThr(CGU)28,50 variants as indicated were grown and analyzed for ORF240 tRNA binding by the pull-down assay as described in C. (E) Schematic of pull-down results in D.