Recruitment of TATA-binding protein-TAF, complex SL1 to the human ribosomal DNA promoter is mediated by the carboxy-terminal activation domain of upstream binding factor (UBF) and is regulated by UBF phosphorylation

Human rRNA synthesis by RNA polymerase I requires at least two auxiliary fa ctors, 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 necess ary for transcription activation and has been shown to be extensively phosp horylated. SL1, which consists of TATA-binding protein (TBP) and three asso ciated factors (TAF(I)s), does not have any sequence-specific DNA binding a ctivity, and its recruitment to the promoter is mediated by specific protei n 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 transcriptiona l 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 acti vation, makes direct contact with the TBP-TAF(1) complex SL1. Since this re gion 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 S L1; moreover, incubation of the dephosphorylated UBF with nuclear extracts from exponentially growing cells was able to restore the UBF-SL1 interactio n. In addition, DNase I footprinting analysis and in vitro-reconstituted tr anscription assays with phosphatase-treated UBF provided further evidence t hat UBF phosphorylation plays a critical role in the regulation of the recr uitment of SL1 to the ribosomal DNA promoter and stimulation of UBF-depende nt transcription.