Comparison of the Effect of Water Release on the Interaction of the Saccharomyces cerevisiae TATA Binding Protein (TBP) with “TATA Box” Sequences Composed of Adenosine or Inosine

Abstract

The formation of sequence-specific complexes of TATA binding protein (TBP) with the minor groove of DNA results in the burial of large nonpolar surfaces and the exclusion of water from these interfaces. The release of water is thus expected to provide a significant entropic driving force for formation of the transcription-preinitiated complexes mediated by the binding of TBP to specific sequences. In this article are described equilibrium-binding studies of Saccharomyces cerevisiae TBP to 14 bp oligonucleotides bearing either the tightly bound and efficiently transcribed adenovirus major late promoter (TATAAAAG) or its inosine-substituted derivative (TITIIIIG) as a function of neutral osmolyte concentration. These two DNA sequences present the same pattern of minor groove hydrogen-bond donors and acceptors to the protein. TBP-DNA complex formation was monitored by steady-state fluorescence resonance energy transfer measurements of the oligonucleotides end-labeled with fluorescein (donor) and TAMRA (acceptor). Correct interpretation of the results obtained with the inosine-substituted sequence required careful consideration of the optical properties of the dyes as a function of osmolyte concentration to demonstrate that the relative change in the end-to-end distances for TATAAAAG- and TITIIIIG-bearing oligonucleotides is the same upon TBP binding. Although the affinity of TBP is slightly greater for the adenosine compared with the inosine-substituted TATA sequence in the absence of osmolyte, the end-to-end distances of the bound DNA in complex with TBP, the enthalpic and electrostatic components of binding, are identical within experimental precision. However, ∼18 additional molecules of water are released upon TBP binding the TATAAAAG as compared with the TITIIIIG sequence resulting in an entropic advantage to the binding of the natural promoter sequence. These results are considered with regard to differences in the flexibility and hydration of the two DNA sequences.