Trm140p (Abp140p) is sufficient for m3C methyltransferase activity. (A) Trm140p is the limiting component for detection of m3C methyltransferase activity in yeast extracts. tRNAThr(IGU) transcribed in vitro with [α-32P] CTP was incubated in methyltransferase buffer containing serial threefold dilutions of extract from wild-type cells (lanes a–c), from trm140-Δ cells (lanes e–g), or from wild-type cells overexpressing frame-fixed Trm140p (Trm140-ff) in which the reading frames of ORFs YOR239W and YOR240W were deliberately fused (lanes i–k) for 2 h at 30°C; then RNA was digested with P1 nuclease; and products were analyzed by chromatography on a PEI-cellulose developed in 0.25 M lithium chloride, as described in Materials and Methods. (m–o) Methyltransferase activity assayed with [α-32P] CTP-transcribed tRNAPhe with the highest concentration of extracts from wild-type cells (m), trm140-Δ cells (n), or wild-type cells overexpressing Trm140p (o). (Lane q) p*m3C control, generated by DMS-treatment of an [α-32P] CTP-labeled tRNAThr(IGU) transcript; (lane r) P*i control. (B) Trm140p expressed and purified from yeast or from E. coli has m3C methyltransferase activity. Methyltransferase activity was examined as described in A, with [α-32P] CTP-transcribed tRNAThr(IGU), using preparations of frame-fixed Trm140p expressed and purified from yeast (lanes a,b), frame-fixed Trm140p expressed and purified from E. coli (d,e), ORF YOR240W (the C-terminal SAM domain) expressed and purified from E. coli (f,g), or with equivalent volumes of a parallel mock purification from E. coli cells containing an empty vector (h,i). c,j, no protein; k–s, assay of methyltransferase using [α-32P] CTP-transcribed tRNAPhe; t, DMS control.
