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Vol. 12, Issue 3, 753-760, March 2001

In Vivo Binding and Hierarchy of Assembly of the Yeast RNA Polymerase I Transcription Factors

Licia Bordi,^* Francesco Cioci, and Giorgio Camilloni

Dipartimento di Genetica e Biologia Molecolare, La Sapienza, Università di Roma, 00185 Rome, Italy

Transcription by RNA polymerase I in Saccharomyces cerevisiae requires a series 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 upstream promoter element, respectively. We have analyzed in vivo the DNAse I footprinting of the 35S promoter in wild-type and mutant strains lacking one specific transcription factor at the time. In this way we were able to unambiguously attribute the protections by the CF and the UAF to their respective putative binding sites. In addition, we have found that in vivo a binding hierarchy exists, the UAF being necessary for CF binding. Because the CF footprinting is lost in mutants lacking a functional RNA polymerase I, we also conclude that the final step of preinitiation-complex assembly affects binding of the CF, stabilizing its contact with DNA. Thus, in vivo, the CF is recruited to the core element by the UAF and stabilized on DNA by the presence of a functional RNA polymerase I.

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^* Present address: I.R.C.C.S. Lazzaro Spallanzani, Via Portuense 292, 00149 Rome, Italy.

Corresponding author. E-mail address: giorgio.camilloni{at}uniroma1.it.

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Molecular Biology of the Cell
Vol. 12, 753-760, March 2001
Copyright © 2001 by The American Society for Cell Biology

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