Transcription Regulation in Pseudomonas aeruginosa

描述（由申请方提供）：铜绿假单胞菌是人类的一种重要机会致病菌，是囊性纤维化（CF）患者发病和死亡的主要原因;铜绿假单胞菌对CF肺的慢性定植通常导致进行性肺损伤，最终导致呼吸衰竭和死亡。拟议的工作将阐明Tex的功能和作用机制，Tex是一种来自铜绿假单胞菌的假定转录因子，在慢性疾病模型中感染宿主肺部所需。Tex在细菌中是高度保守的，并且是真核转录延伸因子Spt 6的同源物。这项研究的基本假设是，特克斯通过影响毒力基因的表达在铜绿假单胞菌的发病机制中起着重要作用，这些毒力基因本身对宿主的感染或存活很重要。为了支持这一观点，初步实验表明，Tex与RNA聚合酶（RNAP），基因表达的中心酶，以及Tex可以影响铜绿假单胞菌中的一个子集的基因的表达。此外，这些实验表明，特克斯不仅与RNAP，但也与一个假定的多亚基复合物的成分，称为RNA降解体：一个核糖核酸酶的复合物首先在E。大肠杆菌，预计主要参与mRNA的降解。这些发现提出了这样的可能性，即在铜绿假单胞菌中，转录机制可能与RNA降解机制有操作上的联系。这样的链接可以想象促进任何必要的处理或降解的特定转录本，并可能代表一种新的机制，引发基因控制。拟议的研究将确定其表达受Tex影响的基因，确定与Tex共纯化的RNA，并测试Tex如何影响基因表达的特定模型。以Tex的高分辨率晶体结构为指导，我们将探索Tex的哪些结构特征对其功能很重要。此外，我们将进一步研究这些蛋白质复合物的性质，含有特克斯和调查的相互作用特克斯与RNAP和组分的推定降解体在铜绿假单胞菌。所提出的实验不仅应该阐明Tex在发病机制中的重要性的分子基础，而且还可以促进可用于对抗CF肺中铜绿假单胞菌感染的新型治疗剂的开发。

DESCRIPTION (provided by applicant): Pseudomonas aeruginosa is an important opportunistic pathogen of humans that is notorious for being the principal cause of morbidity and mortality in Cystic Fibrosis (CF) patients; chronic colonization of the CF lung by P. aeruginosa typically leads to progressive lung damage, and eventually respiratory failure and death. The proposed work will elucidate the function and mechanism of action of Tex, a putative transcription factor from P. aeruginosa that is required for infection of the host lung in a chronic disease model. Tex is highly conserved amongst bacteria, and is a homolog of the eukaryotic transcription elongation factor Spt6. The underlying hypothesis for the proposed study is that Tex plays an important role in P. aeruginosa pathogenesis by influencing the expression of virulence genes that are themselves important for infection of, or survival within, the host. In support of this idea, preliminary experiments indicate that Tex associates with RNA polymerase (RNAP), the central enzyme of gene expression, and that Tex can influence the expression of a subset of genes in P. aeruginosa. Furthermore these experiments reveal that Tex associates not only with RNAP but also with components of a putative multi-subunit complex, termed the RNA degradosome: a ribonuclease-containing complex first described in E. coli that is predicted to be primarily involved in the degradation of mRNA. These findings raise the possibility that the transcription machinery may be operationally linked to the RNA degradation machinery in P. aeruginosa. Such a link could conceivably facilitate any requisite processing or degradation of a particular transcript and may represent a novel mechanism for eliciting gene control. The proposed studies will identify genes whose expression is influenced by Tex, identify RNAs that co-purify with Tex, and test specific models for how Tex might influence gene expression. With a high-resolution crystal structure of Tex as a guide, we will explore which structural features of Tex are important for its function. Moreover, we will investigate further the nature of those protein complexes that contain Tex and investigate the interactions of Tex with both RNAP and components of the putative degradosome in P. aeruginosa. The proposed experiments should not only shed light on the molecular basis for the importance of Tex in pathogenesis, but may also facilitate the development of novel therapeutic agents that can be used to combat P. aeruginosa infection in the CF lung.