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Molecular and Cellular Biology, April 1999, p. 2872-2879, Vol. 19, No. 4
Department of Molecular Microbiology and
Immunology and Norris Comprehensive Cancer Center, University of
Southern California, School of Medicine, Los Angeles, California 90033
Received 22 September 1998/Returned for modification 9 November
1998/Accepted 14 December 1998
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.
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Recruitment of TATA-Binding Protein-TAFI 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
*
Corresponding author. Mailing address: Department of
Molecular Microbiology and Immunology and Norris Comprehensive Cancer Center, University of Southern California, School of Medicine, 2011 Zonal Ave., HMR 509, Los Angeles, CA 90033. Phone: (323) 442-3950. Fax:
(323) 442-1721. E-mail: comai{at}hsc.usc.edu.
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