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Fos-Jun dimerization promotes interaction of the basic region with TFIIE-34 and TFIIF
M L Martin1, P M Lieberman, T Curran
1Roche Institute of Molecular Biology, Nutley, New Jersey 07110, USA.
Abstract:
The regulation of RNA polymerase II-mediated transcription involves both direct and indirect interactions among regulatory proteins and the general transcription factors (GTFs) that assemble at TATA-containing promoters. Here we show that the oncogenic transcription factors Fos and Jun make direct physical contacts with three proteins of the basal transcription apparatus, TFIIE-34 (TFIIE-beta), TFIIF-30 (RAP30), and TFIIF-74 (RAP74). The interactions among the activator proteins and these three GTFs were not detected with other transcription factors, including some bZIP protein family members. Both coimmunoprecipitation and protein blotting experiments demonstrated that the interactions were strongly favored by dimerization of Fos and Jun and that they involved the basic region and basic region-proximal domain of both proteins. Mutations within the DNA-binding domains of Fos and Jun abolished binding to GTFs, although the presence of DNA was not required for the association. Surprisingly, only a single basic region in the context of a protein dimer was sufficient for the interaction. Squelching of AP-1-dependent transcription in vitro by an excess of Fos-Jun dimers was relieved by the addition of TFIIE, indicating that it is a direct functional target of Fos and Jun. These results suggest that dimerization induces a conformational alteration in the basic region of Fos and Jun that promotes an association with TFIIE-34 and TFIIF, thus contributing to transcription initiation.
Insights
Oncogenic transcription factors Fos and Jun directly bind basal transcription factors TFIIE and TFIIF. This interaction, mediated by Fos-Jun dimerization, is crucial for regulating RNA polymerase II transcription initiation.
Area of Science:
- Molecular Biology
- Gene Regulation
- Protein-Protein Interactions
Background:
- RNA polymerase II (Pol II) transcription is a complex process regulated by interactions between transcription factors and general transcription factors (GTFs).
- Oncogenic transcription factors like Fos and Jun play critical roles in cellular processes and disease.
- Understanding the molecular mechanisms of transcription factor interaction with GTFs is essential for deciphering gene regulation.
Purpose of the Study:
- To investigate the direct physical interactions between the oncogenic transcription factors Fos and Jun and components of the basal transcription machinery.
- To elucidate the specific GTFs involved in these interactions and the molecular regions mediating the binding.
- To determine the functional consequences of these interactions on AP-1-dependent transcription.
Main Methods:
- Coimmunoprecipitation assays to detect protein-protein interactions.
- Protein blotting experiments to confirm binding.
- Site-directed mutagenesis to identify critical protein domains involved in binding.
- In vitro transcription assays (squelching) to assess functional impact.
Main Results:
- Fos and Jun directly bind to TFIIE-34 (TFIIE-beta), TFIIF-30 (RAP30), and TFIIF-74 (RAP74).
- These interactions are dependent on Fos-Jun dimerization and involve the basic region and basic region-proximal domain.
- Mutations in the DNA-binding domains of Fos and Jun abolished GTF binding, but DNA was not required for association.
- TFIIE was identified as a direct functional target, as its addition relieved Fos-Jun-mediated squelching of transcription.
Conclusions:
- Dimerization of Fos and Jun induces a conformational change in their basic regions, facilitating association with TFIIE and TFIIF.
- These interactions contribute to the recruitment of basal transcription factors, thereby promoting transcription initiation.
- The findings reveal a novel mechanism by which oncogenic transcription factors directly modulate the transcription machinery.