Rastinejad F, Artz P, Lu P
Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia 19104.
J Mol Biol. 1993 Oct 5;233(3):389-99. doi: 10.1006/jmbi.1993.1519.
The Escherichia coli lac operator DNA contains two sequence repeats related by a pseudo-dyad axis. Deviations from symmetry, in the central 21 bp sequence, occur at two pairs of symmetrically related sites (+15/+7, +13/+9) and at the central base-pair +11. Mutational analysis and DNA protection studies have suggested asymmetric interactions of lac repressor along this sequence. Previous biophysical studies on the lac repressor-operator system have typically employed symmetrized operator sequences to simplify analysis. As a result, it has remained difficult to assess the importance of the naturally occurring sequence deviations from symmetry. Here, 19F-NMR is used to determine if the wild-type E. coli lac operator DNA sequence itself specifies a pair of distinct half-site interactions with lac repressor DNA binding domains. To observe protein interactions simultaneously at operator half-sites using 19F-NMR, three pairs of naturally occurring, symmetry related thymine residues (at +6/+16, +8/+14 and +1/+21) were substituted pair-wise by 5-fluorodeoxyuridines (5-FdU). Two polypeptides corresponding to the N-terminal DNA binding domain of lac repressor "headpiece", residues 1 to 56 and 1 to 64, were employed to remove the steric constraints of subunit interaction in the wild-type tetramer. Spectral changes associated with headpiece binding to left side DNA sequences differ from those caused by binding to equivalent sequences on the right half-site. These results are similar to non-symmetric intact tetramer repressor interactions specified by the DNA sequence. Three mutant lac operator sequences with increased symmetry, bearing FdU substitutions were used to identify the relative importance of the three naturally asymmetric positions. Symmetrizing one pair of these sites alone or in addition to removing the central base-pair failed to produce identical NMR signal changes characteristic of symmetric headpiece-DNA complexes. However, symmetrizing both asymmetric pairs gave chemical shift changes expected from symmetric protein-DNA complexes. We propose that key interactions with the left side +9 (G.C) are altered at the symmetrically related right side +13 (A.T). The data show that the DNA sequence at +13 influences interactions three base-pairs away.
大肠杆菌乳糖操纵基因DNA包含两个由假二元轴相关联的序列重复。在中心21bp序列中，与对称性的偏差出现在两对对称相关的位点（+15/+7，+13/+9）以及中心碱基对+11处。突变分析和DNA保护研究表明，乳糖阻遏物沿着该序列存在不对称相互作用。此前关于乳糖阻遏物-操纵基因系统的生物物理研究通常采用对称化的操纵基因序列以简化分析。因此，评估自然存在的序列对称性偏差的重要性仍然很困难。在此，利用19F-NMR来确定野生型大肠杆菌乳糖操纵基因DNA序列本身是否指定了与乳糖阻遏物DNA结合结构域的一对不同的半位点相互作用。为了使用19F-NMR在操纵基因半位点同时观察蛋白质相互作用，三对自然存在的、对称相关的胸腺嘧啶残基（在+6/+16、+8/+14和+1/+21处）被逐个用5-氟脱氧尿苷（5-FdU）取代。使用了对应于乳糖阻遏物“头部”N端DNA结合结构域的两个多肽，即第1至56位和第1至64位残基，以消除野生型四聚体中亚基相互作用的空间限制。与头部结合到左侧DNA序列相关的光谱变化不同于与结合到右侧半位点等效序列所引起的变化。这些结果类似于由DNA序列指定的非对称完整四聚体阻遏物相互作用。使用了三个具有更高对称性、带有FdU取代的突变乳糖操纵基因序列来确定三个天然不对称位置的相对重要性。单独对称化其中一对位点或除了去除中心碱基对外还对称化其他位点，均未能产生对称头部-DNA复合物特有的相同NMR信号变化。然而，对称化两个不对称对则产生了对称蛋白质-DNA复合物预期的化学位移变化。我们提出，与左侧+9（G.C）的关键相互作用在对称相关的右侧+13（A.T）处发生了改变。数据表明，+13处的DNA序列会影响三个碱基对之外的相互作用。
