Kinetic preference for oriented DNA binding by the yeast TATA-binding protein TBP.
In solution, the TATA box binding protein from S. cerevisiae (yTBP) is only minimally oriented when bound to the adenovirus major late promoter (AdMLP) and the yeast CYC1 promoter. At equilibrium, approximately 60% of the complexes are assembled in the orientation observed within crystal structures; 40% are assembled in the opposite orientation. Here we use stopped-flow fluorescence resonance energy transfer (FRET) to study the association kinetics of the two TBP.TATA box orientational isomers. Kinetics were determined by monitoring FRET between a unique tryptophan residue engineered into either the C- or the N-terminal stirrup of the conserved C-terminal subunit of yeast TBP (yTBPc) and an aminocoumarin moiety appended either upstream or downstream of the TATA box. Together, these constructs permitted a simultaneous yet independent monitor of the kinetics of TBP binding in both orientations. Not only did our results provide an independent confirmation of the free energy difference between the two orientational isomers, but they also showed that the orientational binding preference at equilibrium is a result of a faster association rate when TBP binds DNA in the orientation observed in the crystal structure.
Liu
Y
Y
Department of Chemistry, Yale University, New Haven, Connecticut 06520-8107, USA.
Schepartz
A
A
D003374:Coumarins / Q000378:metabolism; D004271:DNA, Fungal / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019281:Dimerization; D004735:Energy Transfer / Q000235:genetics; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018698:Glutamic Acid / Q000235:genetics; D007700:Kinetics; D008239:Lysine / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D010442:Peptide Chain Initiation, Translational / Q000235:genetics; D011485:Protein Binding / Q000235:genetics; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D016385:TATA Box / Q000235:genetics; D035181:TATA-Box Binding Protein; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014364:Tryptophan / Q000235:genetics
D003374:Coumarins; D004271:DNA, Fungal; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D020033:Protein Isoforms; D035181:TATA-Box Binding Protein; D014157:Transcription Factors; D018698:Glutamic Acid; D014364:Tryptophan; D008239:Lysine; C051029:7-amino-4-methylcoumarin-3-acetic acid