Regulation of expression of the nitrogen fixation (nif) genes of K. pneumoniae

NIFA蛋白（NIFA基因产物）是激活固氮（nif）操纵子转录所必需的，在许多属于革兰氏阴性大分裂变形菌的自由生活和共生细菌中。为了激活NIFA， NIFA结合到nif启动子上游约100 bp的增强子样位点，并允许RNA聚合酶（alpha54-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。我们成功地从肺炎克雷伯菌中纯化了不溶性NIFA蛋白，作为可溶性麦芽糖结合蛋白（MBP）的融合物，并首次在体外证明了其与增强子样位点的结合。MBP-NIFA在纯化的体系中通过α 54-全酶激活转录，并且可以催化该聚合酶和nifH启动子之间的封闭复合物异构化为开放复合物。激活和开放络合物的形成需要具有可水解的β - γ键的三磷酸核苷，但我们无法直接证明水解，因为我们无法从制备中去除污染的水解活性。我们还纯化了一种MBP融合蛋白，在没有dna结合结构域的情况下，只包含NIFA的中心催化结构域，并通过蛋白水解裂解将融合蛋白以可溶性活性形式释放出来。释放的中心结构域可以从溶液中激活转录，这是迄今为止增强子结合蛋白所独有的特性，并且具有水解核苷酸的能力。NIFL蛋白的纯化再生形式，已知在体内对分子氧或组合氮的存在作出反应时抑制NIFA活性，抑制MBP-NIFA和NIFA中心结构域的转录激活。由于NIFL没有通过中心结构域抑制核苷酸水解，我们假设它干扰了NIFA的该结构域和alpha54-全酶之间的蛋白质-蛋白质接触。我们下一个资助期的主要目标是：1)获得具有更好活性的NIFL制剂；2)确定NIFL是否通过化学计量学或共价修饰NIFA来抑制NIFA活性；3)研究NIFL与NIFA、NIFA与聚合酶之间的蛋白-蛋白相互作用；4)确定NIFL感知分子氧的机制。这些研究对于理解增强结合蛋白与靶RNA聚合酶的相互作用以及理解分子氧响应中发生的各种形式的调控具有重要意义。我们希望它们最终能在提高生物固氮效率方面为他人所用。作物的产量常常受到氮的可用性的限制，氮的形式适合于合成蛋白质和生物体的其他大分子特征。某些细菌，无论是单独还是与植物合作，都有能力将大气中的氮气转化为氨，这是一种氮肥，这一过程被称为生物固氮。我们正在研究NIFA蛋白，它是生物固氮的主要调节因子。表征NlFA将有助于了解生物如何在适当条件下解码其DNA的特定部分，并可能有助于提高氮肥的生物产量。下一个资助期的主要目标是了解NIFA蛋白的功能是如何被空气中的氧气毒害的。＊＊＊

9405733 Kustu The NIFA protein (nifA gene product) is required to activate transcription of the nitrogen fixation (nif) operons in a wide variety of free-living and symbiotic bacteria belonging to the large gram-negative division proteobacteria. To activate, NIFA binds to enhancer-like sites approximately 100 bp upstream of nif promoters and allows RNA polymerase (alpha54-holoenzyme form) to denature the DNA strands around a transcriptional startsite -- that is, to isomerize from closed to open complexes. We succeeded in purifying the insoluble NIFA protein from Klebsiella pneumoniae as a fusion to the soluble maltose-binding protein (MBP) and in demonstrating its binding to enhancer-like sites in vitro for the first time. MBP-NIFA activated transcription by alpha54- holoenzyme in a purified system and could be shown to catalyze the isomerization of closed complexes between this polymerase and the nifH promoter to open complexes. Activation and open complex formation required a nucleoside triphosphate with a hydrolyzable, beta-gamma bond but we were unable to demonstrate hydrolysis directly because we could not remove contaminating hydrolytic activities from the preparation. We also purified an MBP fusion to just the central catalytic domain of NIFA, in the absence of its DNA-binding domain, and released the central domain in a soluble, active form by proteolytic cleavage of the fusion protein . The released central domain could activate transcription from solution, a property so far unique among enhancer-binding proteins, and had the expected ability to hydrolyze nucleotides. A purified renatured form of the NIFL protein, which is known to inhibit NIFA activity in vivo in response to the presence of molecular oxygen or combined nitrogen, inhibited transcriptional activation by both MBP-NIFA and the central domain of NIFA. Since NIFL did not inhibit nucleotide hydrolysis by the central domain, we postulate that it interferes with protein-protein contact between this domain of NIFA and alpha54-holoenzyme. Our major goals for the next grant period are: 1) to obtain NIFL preparations with better activity; 2) to determine whether NIFL inhibits NIFA activity by interacting with NIFA stoichiometrically or covalently modifying it; 3) to study protein-protein interactions between NIFL and NIFA and between NIFA and polymerase; and 4) to determine the mechanism by which NIFL senses molecular oxygen. The studies are of interest with respect to understanding the interaction of enhancerbinding proteins with the* target RNA polymerases and with regard to understanding the various forms of regulation that occur in response to molecular oxygen. We hope that they will eventually be of use to others in increasing the efficiency of biological nitrogen fixation. %%% Crop productivity is often limited by the availability of nitrogen in a fornm suitable for synthesis of proteins and other large molecules characteristie of living organisms. (certain bacteria, eithel alone or in partnership with plants, have the ability lo convert nitrogen gas from the atmosphere to ammonia, a nitrogen fertilizer, in a process called biological nitrogen fixation. We are studying the NIFA protein, a major regulator of biological nitrogen fixation. Characterizing NlFA will contnbute to understanding, how organisms decode palticu ar portions of their DNA under appropriate conditions and may help to improve the biological productivn of nitrogen felti1izer. A major goal for the next grant period is to understand how the function of the NIFA protein is poisoned by oxygen gas in the air. ***