Lefevre J F, Lane A N, Jardetzky O
J Mol Biol. 1985 Oct 20;185(4):689-99. doi: 10.1016/0022-2836(85)90054-3.
The dynamic behavior of a palindromic oligonucleotide (C-G-T-A-C-T-A-G-T-T-A-A-C-T-A-G-T-A-C-G) representative of the operator sequence and containing the Pribnow box of the trp operon of Escherichia coli was investigated. The resonances of the imino protons and of the H2 protons of the adenosine residues were all assigned. The opening rate constants of the base-pairs were calculated by monitoring the exchange rate of the observable imino protons (nine out of ten), using selective temperature (T1) measurements, which avoid the complication of cross-relaxation and spin diffusion. These measurements have to be performed in conditions where the exchange process is much faster than the opening and closing of the base-pairs, so that the observed exchange rate is equal to the opening rate. It is shown that the catalysis of the exchange process by phosphate dianions is not very efficient (kB approximately equal to 7 X 10(4) M-1 S-1). Hence, in phosphate buffer, the necessary opening-rate limiting condition is met only at high pH values (approximately equal to 9.5) where efficient catalysis by OH- takes place, or at very high buffer concentration. While G X C base-pairs show very little exchange, acting in the sequence as molecular "staples", the A X T base-pairs that are protected from the fraying have very different opening and closing rates, depending on the sequence. Although it seems possible that the opening process could occur at the base-pair level, it is localized at most to two base-pairs in that particular sequence. The activation energies for the opening process of all non-fraying base-pairs are very similar (19 +/- 1 kcal mol-1; 1 cal = 4.184 J), and the differences in the opening rates are essentially due to differences in the activation entropies. With regard to the role of this sequence in the promoter, it is observed that the end of the Pribnow box exchanges relatively easily, and that the activation parameters for the "breathing" process and for the isomerization step of the promoter--RNA polymerase are not very different.
研究了一种代表操纵序列且包含大肠杆菌色氨酸操纵子Pribnow盒的回文寡核苷酸（C-G-T-A-C-T-A-G-T-T-A-A-C-T-A-G-T-A-C-G）的动态行为。对腺苷残基亚氨基质子和H2质子的共振进行了全归属。通过监测可观测亚氨基质子（十个中的九个）的交换速率，利用选择性温度（T1）测量来计算碱基对的打开速率常数，这种测量避免了交叉弛豫和自旋扩散的复杂性。这些测量必须在交换过程比碱基对的打开和关闭快得多的条件下进行，这样观测到的交换速率就等于打开速率。结果表明，磷酸二阴离子对交换过程的催化效率不高（kB约等于7×10⁴ M⁻¹ s⁻¹）。因此，在磷酸盐缓冲液中，只有在高pH值（约等于9.5，此时OH⁻有高效催化作用）或非常高的缓冲液浓度下，才满足必要的打开速率限制条件。虽然G×C碱基对的交换很少，在序列中起分子“订书钉”的作用，但免受解链影响的A×T碱基对的打开和关闭速率差异很大，这取决于序列。虽然打开过程似乎可能在碱基对水平发生，但在该特定序列中最多局限于两个碱基对。所有不解链碱基对打开过程的活化能非常相似（19±1 kcal mol⁻¹；1 cal = 4.184 J），打开速率的差异主要是由于活化熵的不同。关于该序列在启动子中的作用，观察到Pribnow盒末端相对容易交换，并且启动子- RNA聚合酶“呼吸”过程和异构化步骤的活化参数没有很大差异。
