A conserved region in the σ54‐dependent activator DctD is involved in both binding to RNA polymerase and coupling ATP hydrolysis to activation-猫眼课题宝

σ54 依赖性激活剂 DctD 中的一个保守区域参与与 RNA 聚合酶的结合以及 ATP 水解与激活的耦合

DOI：

发表时间：

1997

期刊：

Molecular Microbiology

影响因子：

3.6

通讯作者：

中科院分区：

生物学2区

文献类型：

作者：

Ying‐Kai Wang;Joon;J. Brewer;T. Hoover

羊根瘤菌DctD通过催化σ 54-RNA聚合酶全酶和启动子之间的封闭复合物异构化为开放复合物来激活dctA启动子的转录。DctD必须与σ 54-全酶进行生产性接触并水解ATP以催化这种异构化。为了进一步定义激活过程，我们试图分离对σ 54-全酶亲和力降低的DctD突变体。突变仅限于蛋白质的高度保守的C3区域，这是偶联ATP水解以在σ 54依赖性激活剂中形成开放复合物所必需的。不能激活转录并且在C3区的C-末端一半具有取代的DctD的突变形式与σ 54和RNA聚合酶的β-亚基有效地交联，表明它们通常与σ 54-全酶结合。相比之下，在交联试验中，C3区N端一半氨基酸取代的DctD的一些突变形式对σ 54和β亚基的亲和力降低。这些数据表明，DctD的C3区的N-末端一半含有在开放复合物形成期间可能接触σ 54-全酶的位点。

Rhizobium melioti DctD activates transcription from the dctA promoter by catalysing the isomerization of closed complexes between σ54‐RNA polymerase holoenzyme and the promoter to open complexes. DctD must make productive contact with σ54‐holoenzyme and hydrolyse ATP to catalyse this isomerization. To define further the activation process, we sought to isolate mutants of DctD that had reduced affinities for σ54‐holoenzyme. Mutagenesis was confined to the well‐conserved C3 region of the protein, which is required for coupling ATP hydrolysis to open complex formation in σ54‐dependent activators. Mutant forms of DctD that failed to activate transcription and had substitutions in the C‐terminal half of the C3 region were efficiently cross‐linked to σ54 and the β‐subunit of RNA polymerase, suggesting that they bound normally to σ54‐holoenzyme. In contrast, some mutant forms of DctD with amino acid substitutions in the N‐terminal half of the C3 region had reduced affinities for σ54 and the β‐subunit in the cross‐linking assay. These data suggest that the N‐terminal half of the C3 region of DctD contains a site that may contact σ54‐holoenzyme during open complex formation.