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J. Biol. Chem., Vol. 277, Issue 37, 34208-34216, September 13, 2002

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The Germ Cell-specific Transcription Factor ALF
STRUCTURAL PROPERTIES AND STABILIZATION OF THE TATA-BINDING PROTEIN (TBP)-DNA COMPLEX^*

Ashok B. Upadhyaya, Mohammed Khan, Tung-Chung Mou, Matt Junker, Donald M. Gray, and Jeff DeJong^§

From the Department of Molecular and Cell Biology, University of Texas at Dallas, Richardson, Texas 75080

The assembly and stability of the RNA polymerase II transcription preinitiation complex on a eukaryotic core promoter involves the effects of TFIIA on the interaction between TATA-binding protein (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 it could not stabilize TBP mutants A184E, N189E, E191R, and R205E nor could it facilitate binding of the TBP-like factor TRF2/TLF to a consensus TATA element. 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 and apparent K_D of this complex was determined to be 650 min 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 TFIIA/ 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 TFIIA/, the TFIIA-dependent interactions of these factors with TBP are nearly indistinguishable in vitro. Thus, a role for ALF in the assembly and stabilization of initiation complexes in germ cells is likely to be similar or identical to the role of TFIIA in somatic cells.

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