Ozoline O N, Uteshev T A, Masulis I S, Kamzolova S G
Institute of Cell Biophysics, Russian Academy of Sciences, Pushchino, Moscow region.
Biochim Biophys Acta. 1993 Mar 20;1172(3):251-61. doi: 10.1016/0167-4781(93)90211-u.
Using a rifampicin-resistant RNA polymerase with altered specificity to different promoters, the D promoter of T7 phage DNA with increased affinity to the mutant enzyme was chosen. This promoter and the T7 A1 promoter with unchanged affinity as well as some nonpromoter DNA fragments were used to compare temperature-induced conformational transitions of RNA polymerase in the course of complex formation. Conformational alterations of RNA polymerase were monitored by the fluorescent label method. It was shown that RNA polymerase undergoes a set of conformational transitions during complex formation with each promoter, some of which were similar by the character of change to spectral parameters of the label (reflecting RPi and, probably, RPo formation). The local structure of complexes formed above 33 degrees C differs for A1 and D. The conformational analysis reveals at least one temperature-dependent stage upon nonspecific interaction of the enzyme with nonpromoter DNA at 13-16 degrees C. Models of functional organization of the enzyme recognizing center and some features of the structure of the promoters which may be essential for their recognition are discussed.
使用对不同启动子具有改变特异性的利福平抗性RNA聚合酶，选择了对突变酶具有更高亲和力的T7噬菌体DNA的D启动子。该启动子和亲和力不变的T7 A1启动子以及一些非启动子DNA片段用于比较在复合物形成过程中RNA聚合酶的温度诱导构象转变。通过荧光标记法监测RNA聚合酶的构象变化。结果表明，RNA聚合酶在与每个启动子形成复合物的过程中经历了一系列构象转变，其中一些转变在变化特征上与标记的光谱参数相似（反映RPi和可能的RPo形成）。在33摄氏度以上形成的A1和D复合物的局部结构不同。构象分析揭示了在13-16摄氏度时酶与非启动子DNA非特异性相互作用时至少一个温度依赖性阶段。讨论了酶识别中心的功能组织模型以及启动子结构中可能对其识别至关重要的一些特征。
