GENE REGULATION IN BACTERIA

细菌中的基因调控

基本信息

批准号：
2174086
负责人：
JOHN R ROTH
金额：
$ 25.67万
依托单位：
UNIVERSITY OF UTAH
依托单位国家：
美国
项目类别：
财政年份：
1977
资助国家：
美国
起止时间：
1977-01-01 至 1998-12-31
项目状态：
已结题

项目摘要

The project proposes an investigation of the control of synthesis, assimilation and recycling of the central metabolic cofactors, NAD and NADP. Emphasis is on pursuing preliminary evidence that cells protect themselves from oxidative stress by reducing their NADH pool. This minimizes production of the damaging -OH radical from H2O2 via the nonenzymatic Fenton reaction, which requires Fe+2 and a reducing agent. NADH, but not NADPH, can serve as the required reducing agent. By reducing NAD levels temporarily, cells can 'freeze' metabolism, allowing time for DNA repair and destruction of H2O2. The model proposes that NAD levels are reduced by conversion of NAD to NADP, followed by destruction of excess NADP. The NMN produced may be excreted to the periplasm for later uptake when growth resumes. The second product of NAD(P) breakdown, 2' 5'ADP, may play a regulatory role in control of the response to oxidative stress. Control of internal iron levels may also be involved in the stress responses; one of the NAD kinases (NAD-more than NADP) has already been shown to be activated by Fe. Other enzymes that initiate the NAD cycle may also be controlled by either O2 or Fe. It is not clear how cells control the relative size of their NAD and NADP pools. We will sequence the two NAD kinase genes, make lac fusions and expression plasmids. Using the fusions, the transcriptional regulation of these genes will be pursued. The purified enzymes will be analysed for modulation of their activity in response to oxidative stress, iron and pyridine precursor limitation. The essential NMN deamidase maintains a low internal level of the toxic intermediate, NMN; we will study the control of this step. Excess NMN may be excreted by the pnuC transporter which appears to contribute to both import and excretion of NMN. The energetics of this transporter will be investigated to determine whether NAD serves to dictate the direction of flow. We will continue the search for transporters of Nm or Na have been described. It seems likely that there must be some control on assimilation of these compound. Several candidates for Nm and Na transporters are in hand which will be pursued to determine how many transporters exist and whether any are controlled. The nadD gene will be tested as a likely candidate for a step that might be regulated to control assimilation and recycling. This compound is a synthetic intermediate, the entry point of assimilated Nm and Na, the entry point of recycled NMN and the source of ribose for synthesis of cobalamin. A missense suppressor will be investigated that may act by unbalancing rNTP pools and increasing the transcriptional error frequency. We pursue this because it is part of a novel idea regarding a possible chemotherapy for AIDS. The idea involves increasing the error rate in transcriptional replication of HIV by attacking host targets, which control purine and pyrimidine pool sizes.

该项目提出了对合成控制的调查，中央代谢辅助因子，NAD和NAD的同化和回收 NADP。重点是追求细胞保护的初步证据通过减少其NADH池来源自氧化应激。这最大程度地减少了通过H2O2的损害-OH生产的产生非酶芬顿反应，需要Fe+2和还原剂。 NADH，但不是NADPH，可以用作所需的还原剂。经过暂时降低NAD水平，细胞可以“冻结”代谢，从而允许 H2O2的DNA修复和破坏的时间。该模型提出了NAD 通过将NAD转换为NADP，降低了水平，然后破坏多余的NADP。产生的NMN可能被排出到周期后来吸收增长时。 NAD的第二个产品（P） 2'5'ADP的崩溃可能在控制中发挥监管作用对氧化应激的反应。内部铁水平的控制也可能参与压力反应； NAD激酶之一（NAD-MORE 比NADP）已经证明是通过Fe激活的。其他酶该启动NAD周期也可以由O2或Fe控制。目前尚不清楚细胞如何控制其NAD的相对大小和 NADP池。我们将对两个NAD激酶基因进行测序，使lac融合和表达质粒。使用融合，转录这些基因的调节将被追求。纯化的酶将是分析了对氧化作用的调节活性的调节应力，铁和吡啶前体的限制。基本NMN Deamidase维持毒性中间体NMN的内部水平较低；我们将研究此步骤的控制。多余的NMN可能被排泄 pNUC转运蛋白似乎有助于进口和 NMN的排泄。该运输蛋白的能量学将是进行了调查以确定NAD是否用于决定流动。我们将继续寻找NM或NA的转运器描述。似乎必须控制这些化合物的同化。 NM和NA的几个候选人运输商在手里进行，以确定多少运输商存在以及是否受到控制。 NADD基因将被视为可能受到监管的步骤的候选人控制同化和回收。该化合物是合成的中间，NM和NA的入口点，入口点回收的NMN和用于合成钴胺的核糖来源。将调查可能通过不平衡采取行动的错义抑制器 RNTP池并增加了转录误差频率。我们追求这是因为它是关于可能的化学疗法的新颖想法的一部分艾滋病。这个想法涉及提高转录的错误率通过攻击宿主目标来复制艾滋病毒，该目标控制着嘌呤和嘧啶池尺寸。