Gallegos M T, Buck M
Department of Biology, Imperial College of Science Technology and Medicine, Sir Alexander Fleming Building, Imperial College Road, London, SW7 2AZ, UK.
J Mol Biol. 1999 May 14;288(4):539-53. doi: 10.1006/jmbi.1999.2704.
Sigma subunits of bacterial RNA polymerases are closely involved in many steps of promoter-specific transcription initiation. Holoenzyme formed with the specialised minor sigma-N (sigmaN) protein binds rare promoters in a transcriptionally inactive state and functions in enhancer-dependent transcription. Using competition and dissociation assays, we show that sigmaN-holoenzyme has a stability comparable to the major sigma70-holoenzyme. Purified partial sequences of sigmaN were prepared and assayed for retention of core RNA polymerase binding activity. Two discrete fragments of sigmaN which both bind the core but with significantly different affinities were identified, demonstrating that the sigmaN interface with core RNA polymerase is extensive. The low affinity segment of sigmaN included region I sequences, an amino terminal domain which mediates activator responsiveness and formation of open promoter complexes. The higher affinity site lies within a 95 residue fragment of region III. We propose that the core to region I contact mediates properties of the sigmaN-holoenzyme important for enhancer responsiveness. Heparin is shown to dissociate sigmaN and core, indicating that disruption of the holoenzyme is involved in the heparin sensitivity of the sigmaN closed complex.
细菌RNA聚合酶的σ亚基密切参与启动子特异性转录起始的多个步骤。由特殊的次要σ-N（σN）蛋白形成的全酶以转录无活性状态结合稀有启动子，并在增强子依赖性转录中发挥作用。通过竞争和解离试验，我们表明σN-全酶的稳定性与主要的σ70-全酶相当。制备了纯化的σN部分序列，并检测其对核心RNA聚合酶结合活性的保留情况。鉴定出σN的两个离散片段，它们都与核心结合，但亲和力明显不同，这表明σN与核心RNA聚合酶的界面很广泛。σN的低亲和力片段包括区域I序列，这是一个氨基末端结构域，介导激活剂反应性和开放启动子复合物的形成。较高亲和力位点位于区域III的一个95个残基的片段内。我们提出，核心与区域I的接触介导了对增强子反应性很重要的σN-全酶的特性。肝素被证明可使σN和核心解离，这表明全酶的破坏与σN封闭复合物的肝素敏感性有关。
