title: The germ cell-specific transcription factor ALF. Structural properties and stabilization of the TATA-binding protein (TBP)-DNA complex.
abstract: 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 TFIIAalpha/beta 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 TFIIAalpha/beta, the TFIIAgamma-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.
authors.affiliation: Department of Molecular and Cell Biology, University of Texas at Dallas, 2601 N. Floyd Road, Richardson, TX 75080, USA.
authors.forename: Ashok B
authors.initials: AB
authors.lastname: Upadhyaya
authors.forename: Mohammed
authors.initials: M
authors.lastname: Khan
authors.forename: Tung-Chung
authors.initials: TC
authors.lastname: Mou
authors.forename: Matt
authors.initials: M
authors.lastname: Junker
authors.forename: Donald M
authors.initials: DM
authors.lastname: Gray
authors.forename: Jeff
authors.initials: J
authors.lastname: DeJong
mesh_terms: D000818:Animals; D002942:Circular Dichroism; D003198:Computer Simulation; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D019281:Dimerization; D005854:Germ Cells / Q000378:metabolism; D051379:Mice; D011401:Promoter Regions, Genetic; D017510:Protein Folding; D017433:Protein Structure, Secondary; D035181:TATA-Box Binding Protein / Q000737:chemistry* / Q000378:metabolism; D035561:Transcription Factor TFIIA / Q000235:genetics; D014157:Transcription Factors / Q000737:chemistry* / Q000502:physiology
chemical_list: C485619:Gtf2a1lf protein, mouse; D035181:TATA-Box Binding Protein; D035561:Transcription Factor TFIIA; D014157:Transcription Factors; D004247:DNA