A minimal RNA polymerase III transcription system

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Abstract
Transcription factor (TF) IIIB recruits RNA polymerase (pol) III for specific initiation of transcription. All three subunits of TFIIIB, TBP, Brf (the TFIIB‐related subunit) and B″, are required for transcription of supercoiled and linear duplex DNA, but we show here that B″ is non‐essential on a promoter that has been partly pre‐opened by unpairing a short segment of the transcription bubble. These findings expose a striking similarity between transcriptional initiation by pol II, pol III and bacterial RNA polymerases: a preformed single‐stranded DNA bubble upstream of the transcriptional start removes the dependence of pol II on TFIIE, TFIIH and ATP hydrolysis, and the dependence of pol III on B″; the favored placement of the transcription bubble for B″‐independent transcription by pol III overlaps a DNA segment that interacts sequence specifically as single‐stranded DNA with the σ70 initiation subunit of Escherichia coli RNA polymerase holoenzyme.