Transcription of Multiple Yeast Ribosomal DNA Genes Requires Targeting of UAF to the Promoter by Uaf30

Abstract

Upstream activating factor (UAF) is a multisubunit complex that functions in the activation of ribosomal DNA (rDNA) transcription by RNA polymerase I (Pol I). Cells lacking the Uaf30 subunit of UAF reduce the rRNA synthesis rate by ∼70% compared to wild-type cells and produce rRNA using both Pol I and Pol II. Miller chromatin spreads demonstrated that even though there is an overall reduction in rRNA synthesis in uaf30 mutants, the active rDNA genes in such strains are overloaded with polymerases. This phenotype was specific to defects in Uaf30, as mutations in other UAF subunits resulted in a complete absence of rDNA genes with high or even modest Pol densities. The lack of Uaf30 prevented UAF from efficiently binding to the rDNA promoter in vivo, leading to an inability to activate a large number of rDNA genes. The relatively few genes that did become activated were highly transcribed, apparently to compensate for the reduced rRNA synthesis capacity. The results show that Uaf30p is a key targeting factor for the UAF complex that facilitates activation of a large proportion of rDNA genes in the tandem array.

SUPPLEMENTAL MATERIAL

Supplemental material for this article may be found at http://mcb.asm.org/ .

ACKNOWLEDGMENTS

We thank the labs of D. T. Auble and M. W. Mayo for access to their PCR machines and antibodies, Mary Bryk for technical advice, Cathleen Josaitis for participation in earlier UAF footprinting with wild-type yeast, and Dan Burke for providing yeast strains. We also thank Yvonne Osheim for critical reading of the manuscript and helpful discussions. Technical assistance with the EM work was provided by Martha Sikes.

This work was supported by NIH grant GM61692 and American Heart Association grants 055490U and 0755633U to J.S.S., NIH grant GM63952 to A.L.B., and NIH grant GM35949 to M.N.