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

肺炎克雷伯菌固氮（nif）基因表达的调节

• 批准号: 9105280
• 负责人: Sydney Kustu
• 金额: $ 30万
• 依托单位: University of California-Berkeley
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-09-01 至 1994-12-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9105280&HistoricalAwards=false
• 关键词: Regulation; Expression; Nitrogen; Fixation; nif

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.

需要nifA基因产物（NIFA蛋白）来激活 固氮（nif）操纵子的转录在广泛的 属于“紫色”门的一种革兰氏阴性细菌 细菌及其亲属”。 为了激活NIFA， nif启动子上游100 bp，允许RNA聚合酶sigma (54全酶形式）以使DNA链变性， 转录起始点-即从封闭异构化为开放异构化 配合物 我们成功地展示了NIFA的活动 克雷伯氏肺炎杆菌蛋白质的体外检测 使我们能够检测到粗制剂中的活性。 我们发现 NIFA在激活转录过程中， 整合宿主因子（IHF），一种使DNA弯曲的小蛋白质 上游NIFA网站和nif发起人之间的合作，以增加合同 在激活剂和聚合酶之间。 在下一次赠款期间 在此期间，我们将尝试纯化活性NIFA并研究其 其DNA结合和推测的ATP酶活性， 在转录激活因子中很少见 此外，我们将使用 用电子显微镜来表征专门的核蛋白 在nif启动子存在下形成的SNUPS结构 激活剂、聚合酶和IHF。 在NIFL在场的情况下 蛋白质，克雷伯氏菌NIFA被分子氧灭活，或 结合氮 我们将试图证明这一点 在粗品制剂中令人信服地灭活，并将其用作 用于纯化和表征NIFL的测定。