Run-off transcription assay with Tfb3ΔC hydrophobic loop (HL) mutants. (A) Run-off transcription with increasing amounts of WT ScTfb3ΔC (lanes 1–5), Tfb3ΔC with HsHL (lanes 6–10), Tfb3ΔC with SpHL (lanes 11–15) and ScRING domain (lanes 16–20). 1.3 pmol of SNR20 98W promoter DNA fragment (–129/+89) was combined with TFIIA (2 pmol), TFIIB (6 pmol), TBP (12 pmol), Sub1 (2 pmol), TFIIE (3 pmol), TFIIF (5.2 pmol), core TFIIH (2 pmol), pol II (5.2 pmol) and Tfb3ΔC or its variants (amounts indicated above each lane). Transcripts initiated from upstream and downstream TSSs are indicated by red and blue arrows, respectively. (B) Same as panel (A) with SNR20 38D promoter DNA fragment (–129/+89). (C) A schematic of SNR20 98W (top) and SNR20 38D (bottom) promoters. TSSs were validated by primer extension analysis (Supplementary Figure S3). TSSs at −7 to −1 may result from slippage synthesis initiated at +1.