SUNY at Albany
June 19-23, 2001
Fluorescence Studies of the Conformations of the N- and C-Domains of Monomeric and Octameric Saccharomyces Cerevisiae TATA Binding Protein (TBP) and Complexed With DNA.
The TATA Binding Protein (TBP) is required for the initiation of eukaryotic gene transcription. The biological function of TBP requires its specific binding to DNA and other transcription factors. Although TBP binds specific sites as a monomer, it oligomerizes in solution to octamers under the experimental conditions of the present studies (1). The crystal structure of the highly conserved C-domain of TBP responsible for the interaction with DNA has been solved alone and complexed to DNA. The N-domain of TBP is variable in length and sequence. Its structure and structural relationship to the C-terminal domain is unknown.
In this study, the intrinsic fluorescence of the six tyrosines located in the C-domain of TBP and the single tryptophan located in the N-domain has been used to monitor the structures of the two domains for TBP monomers, octamers and monomers complexed with a 14 bp oligonucleotide bearing the sequence TATAAAAG. Fluorescence Resonance Energy Transfer (FRET) of a 14-mer double labeled with fluorescein and TAMRA was used to monitor TBP DNA-binding (2). The maximum of the steady-state fluorescence spectrum of TBP is 309 - 310 nm. A normalized difference spectrum of TBP fluorescence with free tyrosine matches the tryptophan fluorescence spectrum of TBP. Thus, the unusually short wavelength maximum of TBP fluorescence is derived not from unusual tryptophan fluorescence, as previously concluded (3), but reflects the unusually high quantum yield of the tyrosine residues in TBP. The tyrosine anisotropy in TBP is very high either in the free protein or complexed with DNA. The tyrosine anisotropy is comparable to that observed in much larger proteins demonstrating rigidity of the C-domain of TBP in monomers, octamers and complexed with DNA. The anisotropy of the single tryptophan within the N-terminal domain of TBP is much lower than that of the tyrosines. This result is consistent with the N-terminal domain being less structured and more mobile than the C-terminal domain (1). Although the fluorescence of tyrosines, but not the tryptophan in TBP is quenched in the complex with DNA, the tryptophan fluorescence is shifted to longer wavelengths in the TBP-DNA complex.
These results show that i) the conformation of the N-domain of TBP in monomers differs of that in octamers (1) while the C-domain structure is unchanged; (ii) the C-domain of TBP has a compact rigid structure that is unaffected by DNA binding; (iii) effective energy migration from the TBP tyrosines to DNA occurs consistent with uniform close proximity; and (iv) the conformation of the N-terminal domain of TBP is flexible and changes upon DNA-binding, a conformational change could impact the subsequent assembly of other transcription factors. Supported by NIH grant GM39929.References and Footnotes
Sergei Khrapunov and Michael Brenowitz
Department of Biochemistry, Albert Einstein College of Medicine,