Single-stranded DNA-binding proteins play many roles in nucleic acid metabolism, but their importance during transcription remains unclear. Quantitative proteomic analysis of RNA polymerase II (RNApII) preinitiation complexes (PICs) identified Sub1 and the replication protein A complex (RPA), both of which bind single-stranded DNA (ssDNA). Sub1, homolog of mammalian coactivator PC4, exhibits strong genetic interactions with factors necessary for promoter melting. Sub1 localizes near the transcription bubble in vitro and binds to promoters in vivo dependent upon PIC assembly. In contrast, RPA localizes to transcribed regions of active genes, strongly correlated with transcribing RNApII but independently of replication. RFA1 interacts genetically with transcription elongation factor genes. Interestingly, RPA levels increase at active promoters in cells carrying a Sub1 deletion or ssDNA-binding mutant, suggesting competition for a common binding site. We propose that Sub1 and RPA interact with the nontemplate strand of RNApII complexes during initiation and elongation, respectively.
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We thank Olga Calvo, Steve Hahn, Richard Kolodner, Michael Hampsey, and Claire Moore for strains and plasmids; Steven Brill for the Rfa1 antibody; Manor Askenazi and Jignesh Parikh for help with mass spectrometry and microarray data analysis; and Minkyu Kim, Sebastian Marquardt, and Nihal Terzi for technical assistance. We also thank Steve Hahn, Michael Meisterernst, Olga Calvo, Alicia Martinez, Doug Koshland, and members of the Buratowski and Marto labs for helpful discussions. This work was supported by NIH grant GM46498 to S.B., a Burroughs Wellcome Fund Career Award in the Biomedical Sciences and NIGMS grant GM079205 to O.J.R., DFCI/Blaise Proteomics Center support to J.M., and a NDSEG predoctoral fellowship to T.S.