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Papers In Press, published online ahead of print July 9, 2002
J. Biol. Chem, 10.1074/jbc.M204808200
Submitted on May 16, 2002
Revised on July 9, 2002
Accepted on July 9, 2002
Department of Molecular and Cellular Biology, University of Texas at Dallas, Richardson, TX 75080
Corresponding Author: dejong{at}utdallas.edu
The assembly and stability of the RNA polymerase II transcription preinitiation complex (PIC) on a eukaryotic core promoter involves the effects of TFIIA on the interaction between TBP and DNA. To extend our understanding of these interactions, we characterized properties of ALF, a germ cell-specific TFIIA-like factor. ALF was able to stabilize the binding of TBP to DNA, but could not stabilize TBP mutants A184E, N189E, E191R and R205E or the TBP-like factor TRF2/TLF. However, phosphorylation of ALF with casein kinase II resulted in the partial restoration of complex formation using mutant TBPs. Studies of ALF-TBP complexes formed on the Adenovirus Major Late (AdML) promoter revealed protection of the TATA box and upstream sequences from -38 to -20 (top strand) and -40 to -22 (bottom strand). The half-life (t1/2) and apparent KD of this complex was determined to be 650 minutes and 4.8 +/- 2.7 nM, respectively. The presence of ALF or TFIIA did not significantly alter the ability of TBP to bind TATA elements from several testis-specific genes. Finally, analysis of the distinct, nonhomologous internal regions of ALF and TFIIAa/b using circular dichroism spectroscopy provided the first evidence to suggest that these domains are unordered, a result consistent with other genetic and biochemical properties. Overall, the results show that while the sequence and regulation of the ALF gene are distinct from its somatic cell counterpart TFIIAa/b, the TFIIAg-dependent interactions of these factors with TBP are nearly indistinguishable in vitro, suggesting that both can fulfill a requirement for stabilizing initiation or reinitiation complexes on the core promoter.
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