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首页> 外文期刊>Journal of bacteriology >In vitro studies of the domains of the nitrogen fixation regulatory protein NIFA.
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In vitro studies of the domains of the nitrogen fixation regulatory protein NIFA.

机译：固氮调节蛋白NIFA结构域的体外研究。

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The prokaryotic enhancer-binding protein NIFA is a multidomain transcriptional activator that catalyzes the formation of open complexes at nitrogen fixation (nif) promoters by a specialized form of RNA polymerase containing sigma 54. The NIFA protein from Klebsiella pneumoniae consists of three domains: the N-terminal domain of unknown function; the central catalytic domain, which is sufficient for transcriptional activation; and the C-terminal DNA-binding domain. Purified fusion proteins between maltose-binding protein (MBP) and NIFA deleted of its N-terminal domain (MBP-delta N-NIFA) or its C-terminal domain (MBP-NIFA-delta C) activated transcription from the K. pneumoniae nifH promoter both in vitro and in vivo. We previously showed that the same was true for a fusion between MBP and the central domain of NIFA. These results indicate that NIFA is sufficiently modular for all fusions carrying its catalytic domain to be active. Unexpectedly, however, simple predictions regarding the location of determinants of the heat lability and insolubility of NIFA, which were based on previous studies of its isolated central and C-terminal domains, were not borne out. Contrary to a previous report from this laboratory, we found that the in vitro start site of transcription for the K. pneumoniae nifH operon could be either of two adjacent G residues, as others had reported in vivo. This was true independent of the activator, i.e., with MBP-NIFA and MBP-delta N-NIFA and with the homologous activator NTRC. When open complexes were formed with GTP as the activating nucleotide, the upstream G residue was probably as a consequence of initiation of transcription.
机译：原核增强子结合蛋白NIFA是一种多域转录激活因子，它通过包含sigma 54的特殊形式的RNA聚合酶催化固氮（nif）启动子上开放复合物的形成。肺炎克雷伯菌的NIFA蛋白由三个域组成：N -功能未知的末端域;中央催化结构域，足以进行转录激活;和C端DNA结合结构域。纯化的麦芽糖结合蛋白（MBP）和NIFA之间的N端结构域（MBP-δN-NIFA）或C端结构域（MBP-NIFA-δC）缺失的融合蛋白激活了肺炎克雷伯菌nifH的转录体内和体外的启动子。先前我们表明，MBP和NIFA中心域之间的融合也是如此。这些结果表明，对于携带其催化结构域的所有融合而言，NIFA具有足够的模块化性。然而，出乎意料的是，没有依据关于NIFA的热不稳定性和不溶性的决定因素的位置进行简单的预测，这些预测是基于先前对其独立的中心和C端结构域的研究得出的。与该实验室先前的报告相反，我们发现肺炎克雷伯菌nifH操纵子的体外转录起始位点可能是两个相邻的G残基之一，正如其他人在体内报道的那样。确实是独立于活化剂的，即与MBP-NIFA和MBP-δN-NIFA以及同源活化剂NTRC无关。当以GTP作为活化核苷酸形成开放复合物时，上游G残基可能是转录起始的结果。

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