Chang B Y, Doi R H
Agricultural Biotechnology Laboratories, National Chung-Hsin University, Taichung, Taiwan, Republic of China.
Biochem J. 1993 Aug 15;294 ( Pt 1)(Pt 1):43-7. doi: 10.1042/bj2940043.
By the use of a partial proteolysis method and Western-blot analysis, the conformational properties of Bacillus subtilis sigma A factor in the transcription initiation stage were studied. From a comparison of the trypsin-digestion patterns of free sigma A and of sigma A associated with core enzyme, it was found that the production of 45 kDa sigma A tryptic-derived fragment was enhanced when sigma A was associated with the core enzyme. More importantly, a 40 kDa sigma A tryptic-derived fragment was found exclusively in this associated state. Based on the change of the digestion kinetics when producing the 45 kDa tryptic fragment and the generation of this new 40 kDa tryptic fragment from sigma A, it was apparent that a conformation change of sigma A occurred during the association of sigma A with the core enzyme. Also, similar patterns were found for the sigma A present in the holoenzyme-promoter DNA complex. These findings suggest that no further distinctive conformational change of sigma A occurs at the step of RNA polymerase holoenzyme and promoter DNA complex formation. Trypsin-digestion patterns of sigma A in different RNA polymerase holoenzyme and promoter DNA complexes were also studied. The presence of similar trypsin digestion-patterns of sigma A in those complexes strongly supports the idea that a similar sigma A conformation is used in the recognition of different sigma A-type promoters and the formation of different open complexes.
通过使用部分蛋白酶解方法和蛋白质免疫印迹分析，研究了枯草芽孢杆菌σA因子在转录起始阶段的构象特性。通过比较游离σA和与核心酶结合的σA的胰蛋白酶消化模式，发现当σA与核心酶结合时，45 kDa的σA胰蛋白酶衍生片段的产生增加。更重要的是，仅在这种结合状态下发现了一个40 kDa的σA胰蛋白酶衍生片段。基于产生45 kDa胰蛋白酶片段时消化动力学的变化以及从σA产生这个新的40 kDa胰蛋白酶片段，很明显在σA与核心酶结合过程中发生了σA的构象变化。此外，在全酶-启动子DNA复合物中存在的σA也发现了类似的模式。这些发现表明，在RNA聚合酶全酶和启动子DNA复合物形成步骤中，σA没有发生进一步明显的构象变化。还研究了不同RNA聚合酶全酶和启动子DNA复合物中σA的胰蛋白酶消化模式。这些复合物中σA存在相似的胰蛋白酶消化模式，有力地支持了这样一种观点，即相似的σA构象用于识别不同的σA型启动子和形成不同的开放复合物。
