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Human rRNA synthesis by RNA polymerase I requires at least two auxiliary factors, upstream binding factor (UBF) and SL1. UBF is a DNA binding protein with multiple HMG domains that binds directly to the CORE and UCE elements of the ribosomal DNA promoter. The carboxy-terminal region of UBF is necessary for transcription activation and has been shown to be extensively phosphorylated. SL1, which consists of TATA-binding protein (TBP) and three associated factors (TAFIs), does not have any sequence-specific DNA binding activity, and its recruitment to the promoter is mediated by specific protein interactions with UBF. Once on the promoter, the SL1 complex makes direct contact with the DNA promoter and directs promoter-specific initiation of transcription. To investigate the mechanism of UBF-dependent transcriptional activation, we first performed protein-protein interaction assays between SL1 and a series of UBF deletion mutants. This analysis indicated that the carboxy-terminal domain of UBF, which is necessary for transcriptional activation, makes direct contact with the TBP-TAFI complex SL1. Since this region of UBF can be phosphorylated, we then tested whether this modification plays a functional role in the interaction with SL1. Alkaline phosphatase treatment of UBF completely abolished the ability of UBF to interact with SL1; moreover, incubation of the dephosphorylated UBF with nuclear extracts from exponentially growing cells was able to restore the UBF-SL1 interaction. In addition, DNase I footprinting analysis and in vitro-reconstituted transcription assays with phosphatase-treated UBF provided further evidence that UBF phosphorylation plays a critical role in the regulation of the recruitment of SL1 to the ribosomal DNA promoter and stimulation of UBF-dependent transcription.