Figure 1.
(A) The single-stranded SNR6 promoter. The 198-nt nontranscribed (Upper) strand comprises the U6 snRNA gene sequence from −60 to +138 relative to the normal transcription start site (bold/underlined; designated as +1). The TATA box and the terminator sequence are in bold letters; the T7-stretch is boxed; 3′ ends of the transcribed strand are indicated by arrows. The extent of the transcription bubble is noted at the bottom. (B) Transcription activity of the single-stranded promoter. Pol III transcription was carried out in the presence of TFIIIB on duplex DNA or 5′ overhang templates, with transcribed-strand 3′ ends located as indicated. Because of the potential inhibitory effect of single-stranded DNA, templates were titrated, as indicated below each lane. (C) Transcriptional activity at 20 fmol 5′ overhang DNA relative to duplex DNA. Error bars are SEM of at least five independent experiments. The distribution of transcriptional start sites, as determined by primer extension, is specified for each construct: open boxes, −2; black, +1; gray, +5. Relative activities of promoters with transcribed strand 3′ ends at base pairs −18, −14, and −12, regarded as indistinguishable from background, were 0.008, 0.008, and 0.009, respectively. (D) TFIIIB dependence of transcription. Transcription was assessed on duplex DNA and the −6 overhang template in the presence of single, any two, or all three TFIIIB subunits. rm, recovery marker.