In vivo binding and hierarchy of assembly of the yeast RNA polymerase I transcription factors

Transcription by RNA polymerase I in Saccharomyces cerevisiae requires a se ries of transcription factors that have been genetically and biochemically identified. In particular, the core factor (CF) and the upstream activation factor (UAF) have been shown in vitro to bind the core element and the ups tream promoter element, respectively. We have analyzed in vivo the DNAse I footprinting of the 35S promoter in wild-type and mutant strains lacking on e specific transcription factor at the time. In this way we were able to un ambiguously attribute the protections by the CF and the UAF to their respec tive putative binding sites. In addition, we have found that in vivo a bind ing hierarchy exists, the UAF being necessary for CF binding. Because the C F footprinting is lost in mutants lacking a functional RNA polymerase I, we also conclude that the final step of preinitiation-complex assembly affect s binding of the CF, stabilizing its contact with DNA. Thus, in vivo, the C F is recruited to the core element by the UAF and stabilized on DNA by the presence of a functional RNA polymerase I.