Regulation of Expression of the Nitrogen Fixation (nif) genes of K. pneumoniae

NIFA基因产物(NIFA蛋白)是激活固氮(NIF)操纵子转录所必需的，这些操纵子属于紫色细菌及其近亲门中的各种革兰氏阴性细菌。为了激活，NIFA结合到nif启动子上游100个碱基上，并允许RNA聚合酶sigma(54全酶形式)变性转录起始点周围的DNA链-即从封闭的复合体异构化为开放的复合体。我们成功地在体外展示了肺炎克雷伯菌NIFA蛋白的活性，开发了一种允许我们检测粗品活性的方法。我们发现NIFA在整合宿主因子(IHF)的帮助下激活转录，IHF是一种小蛋白，可以在上游NIFA位点和NIF启动子之间弯曲DNA，增加激活剂和聚合酶之间的收缩。在下一次资助期间，我们将尝试提纯活性NIFA并研究其性质--其DNA结合和推测的ATPase活性，这在转录激活剂中是罕见的。此外，我们将使用电子显微镜来表征在激活剂、聚合酶和IHF存在下在nif启动子处形成的特殊核蛋白结构(SNUPS)。在Nifl蛋白存在的情况下，Klebsiella Nifa会被分子氧或结合氮灭活。我们将试图在粗制制剂中令人信服地展示这种失活，并将其用作一种分析方法，以纯化和鉴定NIFL。

The nifA gene product (NIFA protein) is required to activate transcription of the nitrogen fixation (nif) operons in a wide variety of gram-negative bacteria belonging to the phylum "Purple bacteria and their relatives". To activate, NIFA binds to sites 100 bp upstream of nif promoters and allows RNA polymerase sigma (54 holoenzyme form) to denature the DNA strands around the transcription startsite- that is, to isomerize from closed to open complexes. We succeeded in demonstrating activity of the NIFA protein from Klebsiella pneumonia in vitro by developing an assay that allowed us to detect activity in crude preparations. We found that NIFA is assisted in activating transcription by the integration host factor (IHF), a small protein that bends the DNA between upstream NIFA sites and nif promoters to increase contracts between the activator and polymerase. During the next grant period, we will attempt to purify active NIFA and study its properties-its DNA-binding and its presumed ATPase activity, which is rare for transcriptional activators. In addition, we will use electron microscopy to characterize specialized nucleoprotein structures (SNUPS) that form at nif promoters in the presence of activator, polymerase, and IHF. In the presence of the NIFL protein, Klebsiella NIFA is inactivated by molecular oxygen or combined nitrogen. We will attempt to demonstrate this inactivation convincingly in crude preparations and, using it as an assay, to purify and characterize NIFL.