The expression of eukaryotic genes is precisely controlled by specific interactions between general transcription initiation factors and gene‐specific transcriptional activators. The general transcription factor TFIID, which plays an essential role in mediating transcriptional activation, is a multisubunit complex comprising the TATA box‐binding protein (TBP) and multiple TBP‐associated factors (TAFs). On the other hand, biochemical and genetic approaches have shown that the promoter‐specific transcriptional activator Sp1 has the ability to interact with one of the components of TFIID, the TBP‐associated factor TAF4. We herein report the structural details of the glutamine‐rich domains (Q‐domains) of Sp1 and TAF4 using circular dichroism (CD) and heteronuclear magnetic resonance (NMR) spectroscopy. We found that the two Q‐domains of Sp1 and four Q‐domains of TAF4 were disordered under physiological conditions. We also quantitatively analyzed the interaction between the Q‐domains of Sp1 and TAF4 by NMR and surface plasmon resonance, and detected a weak but specific association between them. Nevertheless, a detailed analysis of CD spectra suggested that any significant conformational change did not occur concomitantly with this association, at least at the level of the overall secondary structure. These results may represent a prominent and exceptional binding mode for the IDPs, which are not categorized in a well‐accepted concept of “coupled folding and binding.”

An increasing number of proteins, which have neither regular secondary nor well-defined tertiary structures, have been found to be present in cells. The structure of these proteins is highly flexible and disordered under physiological (native) conditions, and they are called "intrinsically disordered" proteins (IDPs). Many of the IDPs are involved in interactions with other biomolecules such as DNA, RNA, carbohydrates, and proteins. While these IDPs are largely unstructured by themselves, marked conformational changes often occur upon binding to an interacting partner, which is known as the "coupled folding and binding mechanism", which enable them to change the conformation to become compatible with the shape of the multiple target biomolecules. We have studied the structure and interaction of eukaryotic transcription factors Sp1 and TAF4, and found that both of them have long intrinsically disordered regions (IDRs). One of the IDRs in Sp1 exhibited homo-oligomer formation. In addition, the same region was used for the interaction with another IDR found in the TAF4 molecule. In both cases, we hay e not detected any significant conformational change in that region, suggesting a prominent and novel binding mode for IDPs/IDRs, which arc not categorized by the well-accepted concept of the coupled folding and binding mechanism.

The promoter‐specific transcription factor Sp1 is expressed ubiquitously, and plays a primary role in the regulation of the expression of many genes. Domains A and B located in the N‐terminal half of the protein are characterized by glutamine‐rich (Q‐rich) sequences. These Q‐rich domains have been shown to be involved in the interaction between Sp1 and different classes of nuclear proteins, such as TATA‐binding protein associated factors. Furthermore, the self‐association of Sp1 via Q‐rich domains is also important for the regulation of transcriptional activity. It has been considered that an Sp1 molecule bound to a “distal” GC‐box synergistically interacts with another Sp1 molecule at a “proximal” binding site. Although the formation of multimers via Q‐rich domains seems functionally important for Sp1, little is known about the structural and physicochemical nature of the interaction between Q‐rich domains. We analyzed the structural details of isolated glutamine‐rich B (QB) domains of Sp1 by circular dichroism (CD), analytical ultracentrifugation, and heteronuclear magnetic resonance spectroscopy (NMR). We found the isolated QB domains to be disordered under all conditions examined. Nevertheless, a detailed analysis of NMR spectra clearly indicated interaction between the domains. In particular, the C‐terminal half was responsible for the self‐association. Furthermore, analytical ultracentrifugation demonstrated weak but significant interaction between isolated QB domains. The self‐association between QB domains would be responsible, at least in part, for the formation of multimers by full‐length Sp1 molecules that has been proposed to occur during transcriptional activation.