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

nifA基因产物（nifA蛋白）是激活各种属于“紫色细菌及其亲戚”门的革兰氏阴性细菌的固氮（nif）操纵子转录所必需的。为了激活NIFA， NIFA结合到nif启动子上游100 bp的位点，并允许RNA聚合酶sigma（54全酶形式）使转录起始位点周围的DNA链变性，即从封闭复合物异构化为开放复合物。我们成功地证明了NIFA蛋白的活性，从克雷伯氏菌肺炎在体外通过开发一种测定，使我们能够检测活性的粗制剂。我们发现NIFA通过整合宿主因子（IHF）来激活转录，整合宿主因子是一种小蛋白质，它使NIFA上游位点和nif启动子之间的DNA弯曲，以增加激活子和聚合酶之间的收缩。在下一个资助期内，我们将尝试纯化活性NIFA并研究其特性-其dna结合和假定的atp酶活性，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，克雷伯菌NIFA被分子氧或组合氮灭活。我们将尝试在粗制剂中令人信服地证明这种失活，并将其用作纯化和表征NIFL的实验。

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.