Sastry S S, Ross B M
Laboratory of Molecular Genetics, Rockefeller University, New York, New York 10021, USA.
Biochemistry. 1996 Dec 10;35(49):15715-25. doi: 10.1021/bi960729d.
Initiation of transcription occurs through a series of steps starting with the binding of RNA polymerase to a promoter DNA and formation of a closed complex. The closed complexes, then isomerize to open complexes. In the open complexes a portion of the promoter DNA is unwound. Using fluorescence spectroscopy, we have investigated in real-time the mechanism of unwinding of promoter DNA during the transition from closed to open complexes of T7 RNA polymerase. We synthesized DNA templates containing the fluorescent base analog 2-aminopurine in place of adenine at specific positions in a T7 RNA polymerase promoter. We located the 2-aminopurine residues in the presumed melting domain of the promoter at -1, -4, and at -6. The fluorescence of 2-aminopurine increases when the DNA goes from a double-stranded form to a single-stranded form. By spectroscopically monitoring the increase in fluorescence of 2-aminopurine in DNA-T7 RNA polymerase complexes, we obtained kinetic and thermodynamic information for DNA unwinding. In the presence of the initiating nucleotide GTP, conformational transitions in the polymerase-promoter complex leading to strand opening were slower than in its absence. The rate of base pair disruption at -1, -6, and at -4 was also slower in the presence of GTP than in its absence. At 37 degrees C, base pair disruption occurred first at -1 followed by -6 and finally at -4. Open complex formation was temperature-sensitive. Temperature effects at -1, -6, and at -4 were consistent with this order of base pair disruption. The apparent activation energies (Ea) for base pair disruption around -1 and -6 were 14 kcal mol-1 and 50 kcal mol-1, respectively, also suggesting this order of base pair disruption. Transcription initiation assays using G-ladder synthesis revealed that initiation rates were almost the same on all three templates containing the modified base. Unlike strand opening, we did not observe lag times for G-ladder synthesis. We suggest that facile base pair disruption at -1 is sufficient for transcription initiation. Based on these data, it is proposed that the polymerase makes contacts at or near -1 and -6 resulting in untwisting of these base pairs thus creating at least two base pair disruption events at -1 and at -6, which are followed by bidirectional propagation to -4.
转录起始通过一系列步骤发生，起始于RNA聚合酶与启动子DNA的结合以及形成闭合复合物。然后，闭合复合物异构化为开放复合物。在开放复合物中，启动子DNA的一部分被解开。我们使用荧光光谱法实时研究了T7 RNA聚合酶从闭合复合物转变为开放复合物过程中启动子DNA解旋的机制。我们合成了在T7 RNA聚合酶启动子的特定位置用荧光碱基类似物2-氨基嘌呤取代腺嘌呤的DNA模板。我们将2-氨基嘌呤残基定位在启动子假定的解链区域的-1、-4和-6位置。当DNA从双链形式转变为单链形式时，2-氨基嘌呤的荧光增强。通过光谱监测DNA-T7 RNA聚合酶复合物中2-氨基嘌呤荧光的增加，我们获得了DNA解旋的动力学和热力学信息。在起始核苷酸GTP存在的情况下，聚合酶-启动子复合物中导致链开放的构象转变比不存在GTP时要慢。在GTP存在的情况下，-1、-6和-4位置的碱基对破坏速率也比不存在GTP时慢。在37摄氏度时，碱基对破坏首先发生在-1位置，随后是-6位置，最后是-4位置。开放复合物的形成对温度敏感。-1、-6和-4位置的温度效应与这种碱基对破坏顺序一致。-1和-6位置附近碱基对破坏的表观活化能（Ea）分别为14千卡/摩尔和50千卡/摩尔，这也表明了这种碱基对破坏顺序。使用G-梯状合成的转录起始测定表明，在所有三个含有修饰碱基的模板上起始速率几乎相同。与链开放不同，我们没有观察到G-梯状合成的延迟时间。我们认为-1位置容易的碱基对破坏足以启动转录。基于这些数据，有人提出聚合酶在-1和-6位置或其附近形成接触，导致这些碱基对解旋，从而在-1和-6位置产生至少两个碱基对破坏事件，随后双向传播至-4位置。
