Härd T, Hsu V, Sayre M H, Geiduschek E P, Appelt K, Kearns D R
Department of Chemistry, University of California at San Diego, La Jolla 92093-0342.
Biochemistry. 1989 Jan 10;28(1):396-406. doi: 10.1021/bi00427a055.
We studied the fluorescence properties of a single tyrosine (Tyr94) located in the C-terminal tail of transcription factor 1 (TF1), a type II procaryotic DNA binding protein encoded by the Bacillus subtilis phage SPO1. The time-resolved fluorescence intensity of Tyr94 in free TF1 dimers decays as a single exponential, and this is consistent with a twofold symmetrical structure. The fluorescence is readily quenched by acrylamide, but it is less accessible to anionic quenchers (iodide and citrate), suggesting that the tyrosine is located on the protein surface in a negatively charged environment provided by neighboring Glu95 and Asp96 residues. TF1 dimers associate at moderate concentrations (greater than 0.02 mg/mL) as judged from concentration dependencies in the molar fluorescence intensity, the steady-state fluorescence polarization, and the bimolecular quenching constants. Nonspecific binding of TF1 to SPO1 and calf thymus (CT) DNA and various double-stranded polynucleotides quenches the Tyr94 fluorescence to varying extent. Fluorescence lifetimes of TF1 in the bound state correlate with spectral overlaps between TF1 emission and DNA absorption, demonstrating that excitation energy transfer to DNA bases contributes significantly to the observed quenching. From analysis of the observed quenching in the DNA complexes we conclude that Tyr94 is located within 10-14 A of the DNA helix axis and not in direct contact with the DNA bases. Equilibrium analyses based on fluorescence titrations show that the maximum binding density on DNA extrapolates to ca. 1 TF1 dimer/5 DNA base pairs. We find several differences in TF1 binding to SPO1 DNA, which contains hydroxymethyluracil instead of thymine, and CT DNA: (i) The tyrosine residue is less exposed to the solvent in the SPO1 DNA complex than in the CT DNA complex. (ii) D2O addition enhances the Tyr94 fluorescence when TF1 binds to SPO1 DNA but not when it binds to CT DNA. (iii) The TF1-SPO1 DNA complex is stable at higher NaC1 concentrations than is the TF1-CT DNA complex, and its formation involves the dissociation of more Na+ ions than does the TF1-CT DNA complex. On the basis of these observations and the fact that the Tyr94-containing tail of TF1 is essential for binding to SPO1 DNA, we discuss various models for the TF1-DNA complex.
我们研究了位于转录因子1（TF1）C末端尾巴上的单个酪氨酸（Tyr94）的荧光特性。TF1是一种由枯草芽孢杆菌噬菌体SPO1编码的II型原核DNA结合蛋白。游离TF1二聚体中Tyr94的时间分辨荧光强度以单指数形式衰减，这与二重对称结构一致。荧光很容易被丙烯酰胺淬灭，但对阴离子淬灭剂（碘化物和柠檬酸盐）的可及性较低，这表明酪氨酸位于由相邻的Glu95和Asp96残基提供的带负电荷环境中的蛋白质表面。根据摩尔荧光强度、稳态荧光偏振和双分子淬灭常数的浓度依赖性判断，TF1二聚体在中等浓度（大于0.02 mg/mL）下会发生缔合。TF1与SPO1和小牛胸腺（CT）DNA以及各种双链多核苷酸的非特异性结合会不同程度地淬灭Tyr94荧光。结合状态下TF1的荧光寿命与TF1发射光谱和DNA吸收光谱之间的光谱重叠相关，表明激发能量向DNA碱基的转移对观察到的淬灭有显著贡献。通过对DNA复合物中观察到的淬灭进行分析，我们得出结论，Tyr94位于距DNA螺旋轴10 - 14 Å范围内，且不与DNA碱基直接接触。基于荧光滴定的平衡分析表明，DNA上的最大结合密度外推至约1个TF1二聚体/5个DNA碱基对。我们发现TF1与含有羟甲基尿嘧啶而非胸腺嘧啶的SPO1 DNA和CT DNA的结合存在几个差异：（i）在SPO1 DNA复合物中，酪氨酸残基比在CT DNA复合物中更少暴露于溶剂中。（ii）添加D2O时，TF1与SPO1 DNA结合时会增强Tyr94荧光，但与CT DNA结合时则不会。（iii）TF1 - SPO1 DNA复合物在比TF1 - CT DNA复合物更高的NaCl浓度下稳定，并且其形成比TF1 - CT DNA复合物涉及更多Na +离子的解离。基于这些观察结果以及TF1含Tyr94的尾巴对于与SPO1 DNA结合至关重要这一事实，我们讨论了TF1 - DNA复合物的各种模型。
