Promoter opening has been analyzed only in the S. cerevisiae (sc) in vitro system. At the sc SUP4 tRNATyr promoter, a DNA segment extending from bp –10 to +7 (relative to the transcriptional start site at +1) opens thermoreversibly between 0 and 40C, the upstream segment, bp –9 to –5, opening at slightly lower temperature than the downstream segment, bp –2 to +7 (Kassavetis et al., 1992). The specification of 17 bp of DNA strand separation at the open promoter could be a slight overestimate. Even under the constraint of attachment to uniquely positioned TFIIIB, pol III can explore a short DNA segment for its initiation site (Fruscoloni et al., 1995). As a consequence, permanganate footprinting, which is used to analyze promoter opening, may provide a snapshot of a frequency-weighted superposition of multiple, closely spaced open promoter complexes that are in dynamic equilibrium instead of the unique complex that is precisely poised to form the first nucleotide linkage. Promoter opening (in linear DNA) naturally involves RNA polymerase, including the initiation-specific C34 subunit (sc gene RPC10) (Brun et al., 1997) and requires the participation of the Brf1 and Bdp1 subunits of sc TFIIIB (Hahn and Roberts, 2000; Kassavetis et al., 1998; Kassavetis et al., 2001). Thus, TFIIIB plays a role in transcriptional initiation that extends beyond recruiting pol III to the promoter.