The Germ Cell-specific Transcription Factor ALF

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 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.Transcription of eukaryotic genes in vitro requires RNA polymerase II and a set of general transcription factors (GTFs)

Expression of the Germ Cell-specific Transcription Factor ALF in Xenopus Oocytes Compensates for Translational Inactivation of the Somatic Factor TFIIA

“…The extracts were centrifuged at 14,000 rpm for 15 min to obtain clarified lysates. TBP-dependent mobility shift assays were performed as described (7).…”

“…Earlier results had suggested that ALF and TFIIA were similar with respect to their ability to interact with TBP (7) and that both could support RNA polymerase II activity in vitro (5,6). Even though TFIIA is not normally present in oocytes, we wanted to evaluate whether it could function in this system.…”

“…Previous studies have shown that the expression of ALF is restricted to male germ cells in the testis of mice and humans (5,6) and that it is up-regulated during the pachytene stage of meiosis I along with a number of other transcription factors (8). However, biochemical characterization of recombinant ALF-TFIIA␥ and TFIIA␣/␤-TFIIA␥ complexes did not reveal significant differences in vitro (7), and it remains unclear what specific role ALF might play during germ cell development.…”

“…This factor is a counterpart of the large (␣/␤) subunit of TFIIA. Like TFIIA␣/␤, ALF is able to interact with the small TFIIA subunit (␥) to form a heterodimeric complex that stabilizes binding of TBP to promoter DNA (7). Previous studies have shown that the expression of ALF is restricted to male germ cells in the testis of mice and humans (5,6) and that it is up-regulated during the pachytene stage of meiosis I along with a number of other transcription factors (8).…”

Regulatory Factor Interactions and Somatic Silencing of the Germ Cell-specific ALF Gene

“…One of these germ cell variants, TFIIA␣/␤-like factor (ALF) 2 (TFIIA), encodes a counterpart of the large subunit of the general transcription factor TFIIA (4 -6). ALF interacts with the small TFIIA␥ subunit to form a complex that can stabilize binding of TBP-like factors to promoter DNA, thereby facilitating formation of a transcription preinitiation complex in germ cells (7,8). The loss of ALF expression is associated with male infertility in humans (9).…”