Kinetic Preference for Oriented DNA Binding by the Yeast TATA-Binding Protein TBP

详细信息    查看全文

• 作者：Yichin Liu and Alanna Schepartz
• 刊名：Biochemistry
• 出版年：2001
• 出版时间：May 29, 2001
• 年：2001
• 卷：40
• 期：21
• 页码：6257 - 6266
• 全文大小：610K
• 年卷期：v.40,no.21(May 29, 2001)
• ISSN：1520-4995

文摘

In solution, the TATA box binding protein from S. cerevisiae (yTBP) is only minimally orientedwhen 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 energytransfer (FRET) to study the association kinetics of the two TBP·TATA box orientational isomers. Kineticswere 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 aminocoumarinmoiety appended either upstream or downstream of the TATA box. Together, these constructs permitteda simultaneous yet independent monitor of the kinetics of TBP binding in both orientations. Not only didour results provide an independent confirmation of the free energy difference between the two orientationalisomers, but they also showed that the orientational binding preference at equilibrium is a result of afaster association rate when TBP binds DNA in the orientation observed in the crystal structure.