Mechanism of heme regulation of a P. aeruginosa non-coding RNA

描述（由申请方提供）：铜绿假单胞菌是一种机会致病菌，可导致受损个体严重感染，包括血小板减少性癌症患者和囊性纤维化（CF）患者。为了建立成功的感染，铜绿假单胞菌需要铁，并采用几种策略来获得铁，包括摄取血红素。虽然需要生存，过剩的铁或血红素可能会导致氧化应激;因此，铁吸收调节（Fur）蛋白调节这些营养素的吸收。在充满铁的环境中，Fur阻断铁和血红素摄取所需的基因以及编码PrrF 1和PrrF 2小调控RNA的两个几乎相同的基因的表达（Wilderman等人，2004年）。我们先前表明PrrF RNA负面影响至少50个基因的表达，这些基因的产物在关键代谢途径中起作用，并且其中一个深刻地影响群体感应因子的产生（奥格尔斯比等人，2008年）。因此，PrrF RNA能够对铜绿假单胞菌的生理学和毒力产生广泛的影响。血红素是人体中丰富的铁来源，并且其被铜绿假单胞菌获得被假设在感染中起重要作用。由于血红素的潜在毒性作用，预期血红素调节系统协调血红素摄取、降解和生物合成的基因表达。由于血红素对毒力的实质性影响，确定这种化合物作为信号分子的机制将产生用于治疗目的的新靶点。铜绿假单胞菌中的PrrF RNA由两个几乎相同的基因prrF 1和prrF 2串联编码（图1），而所有其他假单胞菌在远端基因座编码PrrF RNA。I和Vasil博士实验室中的其他人表明，铜绿假单胞菌的prrF基因座编码另外的325个核苷酸（nt）RNA，称为PrrH，其被血红素以及铁抑制（Oglesby-Sherrouse & Vasil，2010，Ochsner et al.，2000年）。PrrH的转录起始于prrF 1的5'端，通过prrF 1-prrF 2基因间序列（95 nt）进行，并终止于prrF 2的3'端（图1）。因此，prrH的表达依赖于prrF 1 Rho非依赖性或内在终止子处的通读转录。我的数据表明，PrrH调节参与血红素生物合成（奥格尔斯比- Sherrouse & Vasil，2010）和毒力（表1）的基因，表明这种串联prrF组织赋予铜绿假单胞菌独特的血红素调节活性。我假设血红素调节的抗终止子（PHAT -图1）识别prrF 1终止子之前的RNA序列或之后的DNA序列，并解除新生RNA的发夹结构，从而使转录延伸复合物（TEC）稳定并使PrrH RNA继续转录。该提案将通过以下方式确定prrH表达和血红素调节的机制：I）确定prrH和推定的PrrH靶标的表达和血红素调节的序列要求，以及II）鉴定参与prrH表达和调节的基因和蛋白质。

DESCRIPTION (provided by applicant): Pseudomonas aeruginosa is an opportunistic pathogen that causes severe infection in compromised individuals, including neutropenic cancer patients and individuals with cystic fibrosis (CF). To establish successful infection, P. aeruginosa requires iron and employs several strategies for its acquisition, including the uptake of heme. Although required for survival, surplus iron or heme can lead to oxidative stress; thus, the ferric uptake regulator (Fur) protein regulates the uptake of these nutrients. In iron-replete environments, Fur blocks expression of genes required for iron and heme uptake, as well as two nearly identical genes encoding the PrrF1 and PrrF2 small regulatory RNAs (Wilderman et al., 2004). We previously showed that the PrrF RNAs negatively affect the expression of at least 50 genes, the products of which function in key metabolic pathways, and one of which profoundly affects production of quorum sensing factors (Oglesby et al., 2008). Consequently, the PrrF RNAs are capable of exerting wide-ranging effects on P. aeruginosa physiology and virulence. Heme is an abundant source of iron in the human body, and its acquisition by P. aeruginosa is hypothesized to play a significant role in infection. Because of the potentially toxic effects of heme, it is expected that a heme regulatory system coordinates expression of genes for heme uptake, degradation, and biosynthesis. With the substantial implications that heme has for virulence, identifying the mechanism(s) by which this compound acts as a signaling molecule will yield novel targets for therapeutic purposes. The PrrF RNAs in P. aeruginosa are encoded in tandem by two virtually identical genes, prrF1 and prrF2 (Fig. 1), while all other pseudomonads encode for the PrrF RNAs at distal loci. I and others in Dr. Vasil's laboratory showed that the prrF locus of P. aeruginosa encodes an additional, 325-nucleotide (nt) RNA, designated PrrH, which is repressed by heme as well as iron (Oglesby-Sherrouse & Vasil, 2010, Ochsner et al., 2000). Transcription of PrrH initiates at the 5' end of prrF1, proceeds through the prrF1-prrF2 intergenic sequence (95 nt), and terminates at the 3' end of prrF2 (Fig. 1). Thus, expression of prrH is dependent on read-through transcription at the prrF1 Rho-independent, or intrinsic, terminator. My data indicate PrrH regulates genes involved in heme biosynthesis (Oglesby- Sherrouse & Vasil, 2010) and virulence (Table 1), suggesting this tandem prrF organization imparts unique heme regulatory activities to P. aeruginosa. I hypothesize that a heme- regulated antiterminator (PHAT - Fig. 1) recognizes RNA sequence preceding or DNA sequence following the prrF1 terminator and relieves the hairpin structure of the nascent RNA, allowing stabilization of the transcription elongation complex (TEC) and continued transcription of the PrrH RNA. This proposal will determine the mechanism of prrH expression and heme regulation by I) determining the sequence requirements for expression and heme regulation of prrH and putative PrrH targets and II) identifying genes and proteins involved in expression and regulation of prrH.