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首页> 外文期刊>Journal of bacteriology >Alterations within the activation domain of the sigma 54-dependent activator DctD that prevent transcriptional activation.
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Alterations within the activation domain of the sigma 54-dependent activator DctD that prevent transcriptional activation.

机译：σ54依赖性激活子DctD的激活域内的变化可防止转录激活。

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摘要

Rhizobium meliloti DctD (C4-dicarboxylate transport protein D) is a transcriptional activator that catalyzes the ATP-dependent isomerization of closed complexes between sigma 54-RNA polymerase holoenzyme and the dctA promoter to open complexes. Following random mutagenesis of dctD, 55 independent mutant forms of DctD that failed to activate transcription from a dctA'-'lacZ reporter gene in Escherichia coli were selected, and the amino acid substitutions were determined for these mutant proteins. Amino acid substitutions were distributed throughout the central domain of the protein, the domain responsible for transcription activation, but most of the substitutions occurred within three highly conserved regions of the protein. Selected mutant proteins were purified, and their activities were studied in vitro. All of the purified mutant proteins appeared to have normal DNA-binding activity and interacted with sigma 54 and core RNA polymerase, as determined from protein crosslinking assays. Proteins with amino acid substitutions in a region spanning amino acid positions 222 to 225 retained their ATPase activities, whereas proteins with substitutions in other regions had little or no ATPase activity. Taken together, these data suggest that the region that encompasses amino acid residues 222 through 225 probably functions in coupling the energy released from ATP hydrolysis to open complex formation rather than as a major determinant for binding to RNA polymerase.
机译：苜蓿根瘤菌DctD（C4-二羧酸转运蛋白D）是一种转录激活剂，可催化σ-54-RNA聚合酶全酶和dctA启动子之间的闭合复合物的ATP依赖性异构化，从而打开复合物。在随机诱变dctD之后，选择了55种独立的突变形式的DctD，它们未能激活大肠杆菌中dctA'-'lacZ报告基因的转录，并确定了这些突变蛋白的氨基酸取代。氨基酸取代分布在蛋白质的整个中央结构域（负责转录激活的结构域）中，但是大多数取代发生在蛋白质的三个高度保守的区域内。纯化选定的突变蛋白，并在体外研究其活性。所有纯化的突变蛋白似乎都具有正常的DNA结合活性，并与sigma 54和核心RNA聚合酶相互作用，这是通过蛋白质交联测定确定的。在跨氨基酸位置222至225的区域中被氨基酸取代的蛋白质保留了其ATPase活性，而在其他区域中被取代的蛋白质则几乎没有或没有ATPase活性。综上，这些数据表明，包含氨基酸残基222至225的区域可能起着将ATP水解释放的能量耦合至开放复合物形成的作用，而不是作为与RNA聚合酶结合的主要决定因素。

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