FIGURE 1.
Maf1 represses multiple round but not single round transcription by human RNA pol III. A, scheme to test the effect of Maf1 on HeLa nuclear extract (NE)-supported tRNALeu3 gene transcription. NE was preincubated with Maf1 or BSA for 10 min at room temperature. Cold nucleotides, [α-32P]CTP (C*), and plasmid tRNALeu3 template (0.5 μg) were added, and transcription was allowed to proceed for 50 min at 30 °C. B, lanes 1–6 show transcription supplemented with increasing amounts of recombinant human Maf1, (0–0.5 μg/reaction). Lane 7 contained NE with BSA (0.5 μg) as a negative control. C, the data in B were quantified and plotted relative to the unsupplemented NE. Rel Txn, relative transcription. D, as outlined, after preincubation of the plasmid template and NE, Maf1, or Sarkosyl and nucleotides were added, and the reactions were stopped after the times indicated. E, lanes 1–4 represent conditions that allow multiple round transcription (MR); lanes 5–8 were supplemented with Maf1 (0.5 μg); and lanes 9–12 contained Sarkosyl at a final concentration of 0.05% (SR Sark). F, transcript levels from E were quantified relative to the Sarkosyl-treated reaction (E, lane 9). Open bars, MR transcription; filled bars, Maf1-repressed transcription; shaded bars, SR transcription. One microgram of recombinant hMaf1/50-μl reaction corresponds to a concentration of 0.7 μm.