Abstract
The accurate transcription of human rRNA genes by RNA polymerase I requires two transcription factors, upstream binding factor (UBF) and promoter selectivity factor (SL1). Human SL1 (hSL1) is a multisubunit complex, one of whose components is TATA box-binding protein (TBP). hSL1 binds to the core region of the rRNA promoter, but does so inefficiently in the absence of human UBF (hUBF). hUBF interacts with the upstream control element of the rRNA promoter and facilitates binding of hSL1. The molecular basis by which hUBF increases binding of hSL1 remains to be elucidated. In this report, we use an immobilized protein binding assay to identify and purify a 95-kDa TBP-binding polypeptide. Microsequence analysis reveals that the 95-kDa TBP-binding protein is hUBF. We show that hUBF is stably associated with TBP and is present in large TBP-containing complexes. Our results indicate that the cooperative binding of hUBF and hSL1 on the rRNA promoter is mediated by direct interaction between hUBF and TBP. We also provide evidence that hUBF associates with TFIID, a TBP-containing RNA polymerase II transcription factor.
Highlights
Upstream binding factor (UBF) and promsoetleerctivity Here we report that a TBP-binding protein identified by a factor (SL1).Human SL1 is a multisubunit com- membrane-immobilized protein binding assay is hUBF
The plex, oneof whose componentsis TATAbox-binding pro- direct interaction between hUBF and TBP explains the basis tein (TBP). hSLl binds to the core region of the rRNA for their cooperative binding to the rRNA promoter
But doesso inefficiently in the absenceof hu- find,unexpectedly, that an antibody against hUBF inhibits manUBF. hUBF interacts with the upstream RNA polymerase 11-directed transcriptional activation by sevcontrol element of the rRNA promoter and facilitates eral upstream activatorbsu, t not an activator-independent“babinding of Human SL1 (hSL1)
Summary
Upstream binding factor (UBF) and promsoetleerctivity Here we report that a TBP-binding protein identified by a factor (SL1).Human SL1 (hSL1) is a multisubunit com- membrane-immobilized protein binding assay is hUBF. These and other results indicate that hUBF is associated with TFIID, a TBP-containing RNA polymerase I1 transcription factor. Protein Purification-The 95-kDa TBP-bindingprotein was purified fromHeLacell nuclear pellets recoveredfollowing preparation of a standard nuclear extract [13].Two volumesof BufferA(50mM Tris-HCI, The human RNA polymerase I transcriptionfactor SL1 5 mM MgCl,, 5 mM DTT, 0.5 mM EDTA,0.2 m~ phenylmethylsulfonyl (hSLl), required for transcription of human rRNA genes, is composed of TBP and three TAFs [1].
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