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

9405733 Kustu NIFA蛋白（nifA基因产物）是激活属于大型革兰氏阴性分裂变形菌的多种自由生活和共生细菌中固氮（nif）操纵子转录所必需的。为了激活，NIFA与nif启动子上游约100 bp的增强子样位点结合，并允许RNA聚合酶（α 54全酶形式）使转录起始位点周围的DNA链变性，即从封闭的复合物异构化为开放的复合物。我们成功地纯化了不溶性NIFA蛋白从肺炎克雷伯氏菌作为融合的可溶性麦芽糖结合蛋白（MBP），并在体外首次证明其结合到增强子样位点。MBP-NIFA在纯化的系统中通过α 54全酶激活转录，并且可以显示出催化该聚合酶和nifH启动子之间的封闭复合物异构化为开放复合物。活化和开放复合物的形成需要具有可水解的β-γ键的核苷三磷酸，但我们无法直接证明水解，因为我们无法从制剂中去除污染的水解活性。我们还纯化了MBP融合到NIFA的中心催化结构域，在其DNA结合结构域的情况下，并通过融合蛋白的蛋白水解切割以可溶性活性形式释放中心结构域。释放的中央结构域可以激活转录从溶液中，到目前为止，增强子结合蛋白中的独特的属性，并具有预期的能力，水解核苷酸。 已知NIFL蛋白的纯化复性形式在体内响应于分子氧或结合氮的存在而抑制NIFA活性，其通过MBP-NIFA和NIFA的中心结构域抑制转录激活。由于NIFL不抑制核苷酸水解的中心结构域，我们推测，它干扰蛋白质之间的接触NIFA和α 54全酶的这个结构域。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是否通过与NIFA化学计量或共价修饰相互作用来抑制NIFA活性; 3）研究NIFL与NIFA之间以及NIFA与聚合酶之间的蛋白质-蛋白质相互作用;以及4）确定NIFL感知分子氧的机制。这些研究对于理解增强子结合蛋白与靶RNA聚合酶的相互作用以及理解分子氧反应中发生的各种形式的调节是有意义的。我们希望它们最终能在提高生物固氮效率方面对其他人有用。 农作物的生产力通常受到氮素有效性的限制，氮素的形式适合于蛋白质和其他生物体所特有的大分子的合成。（某些细菌，无论是单独的还是与植物合作的，都具有在称为生物固氮的过程中将大气中的氮气转化为氨（一种氮肥）的能力。 我们正在研究NIFA蛋白，它是生物固氮的主要调节剂。对NlFA的研究将有助于理解生物体在适当条件下如何解码其DNA的触觉部分，并可能有助于提高氮肥的生物生产力。下一个资助期的一个主要目标是了解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. ***