Functional Silencing of TATA-binding Protein (TBP) by a Covalent Linkage of the N-terminal Domain of TBP-associated Factor 1

Abstract

General transcription factor TFIID is comprised of TATAbinding protein (TBP) and TBP-associated factors (TAFs), together playing critical roles in regulation of transcription initiation. The TAF N-terminal domain (TAND) of yeast TAF1 containing two subdomains, TAND1 (residues 10 -37) and TAND2 (residues 46 -71), is sufficient to interact with TBP and suppress the TATA binding activity of TBP. However, the detailed structural analysis of the complex between yeast TBP and TAND12 (residues 6 -71) was hindered by its poor solubility and stability in solution. Here we report a molecular engineering approach where the N terminus of TBP is fused to the C terminus of TAND12 via linkers of various lengths containing (GGGS) n sequence, (n ‫؍‬ 1, 2, 3). The length of the linker within the TAND12-TBP fusion has a significant effect on solubility and stability (SAS). The construct with (GGGS) 3 linker produces the best quality single-quantum-coherence (HSQC) NMR spectrum with markedly improved SAS. In parallel to these observations, the TAND12-TBP fusion exhibits marked reduction of TBP function in binding to TAF1 as well as temperature sensitivity in in vivo yeast cell growth. Remarkably, the temperature sensitivity was proportional to the length of the linker in the fusions: the construct with (GGGS) 3 linker did not grow at 20°C, while those with (GGGS) 1 and (GGGS) 2 linkers did. These results together indicate that the native interaction between TBP and TAND12 is well maintained in the TAND12-(GGGS) 3 -TBP fusion and that this fusion approach provides an excellent model system to investigate the structural detail of the TBP-TAF1 interaction.

Solution structures of yeast Saccharomyces cerevisiae calmodulin in calcium‐ and target peptide‐bound states reveal similarities and differences to vertebrate calmodulin

“…Subsequent NMR structural studies using a single‐chain chimeric protein mimicking a protein–ligand complex have been reported (Candel et al. 2007; Mal et al. 2007).…”

“…Structure of the complex in the Ca 2+ -bound state Porumb et al (1994) initially showed that an engineered chimeric protein of calmodulin and the M13peptide appears to have high thermal stability and high binding affinity for Ca 2+ ions. Subsequent NMR structural studies using a single-chain chimeric protein mimicking a protein-ligand complex have been reported (Candel et al 2007;Mal et al 2007). Advantages of the chimeric protein for structural analysis include increased binding affinity of lowaffinity ligands and stabilization of protein-ligand complexes for long-term NMR measurements.…”

Recognition of the disordered p53 transactivation domain by the transcriptional adapter zinc finger domains of CREB-binding protein

“…To circumvent this problem, we created fusion proteins, in which the two interacting partners are joined by a flexible linker. Fusion proteins enforce close proximity of the components of the complex, increasing the effective concentration and association rate and driving the binding process into the slow-exchange regime, thereby reducing line broadening and enhancing spectral quality (55,56). On the basis of the preliminary structure of the nontethered complex, we designed a fusion protein containing the TAZ2 domain of mouse CBP (residues 1764-1855) joined to the p53TAD (residues 2-61) by a six-residue Gly-Ser repeat sequence (Fig.…”

Overcoming the solubility limit with solubility-enhancement tags: successful applications in biomolecular NMR studies

“…It should be noted that an "active" fusion tag can also be highly effective. For example, Ikura and co-workers fused the TAF N-terminal Domain 1 and 2 (TAND12) with its binding partner TATA-binding protein (TBP) to form a stable protein complex, which displayed enhanced solubility and sample stability (Mal et al 2007). However, such an "active" fusion tag is target specific and cannot be easily applied to other proteins.…”