Comparative Analysis of RNA Helicases in E. coli

描述(申请人提供)：DEAD-box RNA解旋酶是一类功能广泛的保守蛋白质，其特征是能够解开RNA双链，被认为在调节RNA功能中发挥重要作用。然而，许多DEAD-box蛋白的研究很少，因此它们的细胞功能尚不清楚。如果没有这些知识，对RNA新陈代谢的详细了解仍然是不完整的。我们建议研究两个大肠杆菌DEAD-box RNA解旋酶，srmB和DEAD，它们与RNA代谢的不同方面有关。我们的初步数据表明：(I)srmB和DEAD各自促进体内非进行性外源核糖核酸酶--多核苷酸磷酸化酶(PNPase)消化结构化RNA；(Ii)缺乏这两个因子导致8%的大肠杆菌转录产物的差异调节，可能是通过影响RNA的构象和周转；(Iii)srmB和死亡菌株的冷敏感生长表型可以被过表达的核糖体RNA加工和修饰因子抑制；(Iv)大肠杆菌死盒螺旋酶刺激聚(A)聚合酶(PAP)的功能，PAP是一种使细胞转录物多腺化并针对其降解的酶。因此，这些蛋白质中的每一个在RNA周转中都有多种作用。结合使用遗传和生化方法，拟议研究的重点将是详细定义这些功能。首先，我们将进行全基因组调查，以确定通过srmB发生翻转或PNPase功能的死亡刺激的细胞转录本。我们还将对影响这些特性的RNA决定因素进行生化表征。其次，我们将鉴定与srmB直接相互作用或在细胞中死亡的转录本，并研究这些因素对差异转录的调控机制。第三，我们将研究核糖体RNA加工因子的过度表达抑制srmB和死亡冷敏感生长缺陷的基础。第四，我们将分析死盒螺旋酶刺激PAP的机制和后果。总体而言，这些研究应该为靶标选择的基础、与RNA加工酶的功能相互作用以及两个重要的DEAD-BOX蛋白的细胞功能提供见解。这样的发现应该与尚未详细描述的死盒蛋白广泛相关。项目叙事 通过提供对RNA代谢的更多了解，对死盒RNA解旋酶的拟议研究将为更好地了解由于RNA功能缺陷引起的疾病提供更好的见解。对这类RNA解旋酶的先进知识也可能有助于设计针对病原生物体中保守的死盒结构域的新型抗生素。

DESCRIPTION (provided by applicant): DEAD-box RNA helicases are a versatile class of conserved proteins characterized by their ability to unwind RNA duplexes, which are believed to play important roles in regulating RNA function. However, many DEAD- box proteins have been poorly studied and hence their cellular functions are unknown. Without this knowledge, a detailed understanding of RNA metabolism remains incomplete. We propose to study two E. coli DEAD-box RNA helicases, SrmB and DeaD, which have been implicated in different aspects of RNA metabolism. Our preliminary data indicate the following: (i) SrmB and DeaD each promote a non-processive exo-ribonuclease, polynucleotide phosphorylase (PNPase), to digest structured RNA in vivo; (ii) the absence of either factor results in differential regulation of 8% of E. coli transcripts, presumably through an effect on RNA conformation and turnover; (iii) the cold-sensitive growth phenotype of srmB and deaD strains can be suppressed by over-expressing ribosomal RNA processing and modification factors; (iv) E. coli DEAD-box helicases stimulate the function of poly(A) polymerase (PAP), an enzyme that polyadenylates cellular transcripts and targets them for degradation. Therefore, each of these proteins has multiple roles in RNA turnover. Using a combination of genetic and biochemical approaches, the focus of the proposed studies will be to define these functions in detail. First, we will conduct a genome-wide survey to identify cellular transcripts that undergo turnover via SrmB or DeaD stimulation of PNPase function. We will also biochemically characterize the RNA determinants that are responsible for these properties. Second, we will identify transcripts that directly interact with SrmB or DeaD in the cell, and investigate the mechanism of differential transcript regulation by these factors. Third, we will investigate the basis for suppression of the srmB and deaD cold-sensitive growth defect due to the over-expression of ribosomal RNA processing factors. Fourth, we will analyze the mechanism and consequences of PAP stimulation by DEAD-box helicases. Overall, these studies should provide insights into the basis of target selection, functional interactions with RNA processing enzymes and the cellular functions of two important DEAD-box proteins. Such findings should be broadly relevant to DEAD-box proteins that have yet to be characterized in detail. PROJECT NARRATIVE By providing a greater understanding of RNA metabolism, the proposed studies on DEAD-box RNA helicases will provide better insights into diseases that are due to defects in RNA function. Advanced knowledge of such RNA helicases could also be helpful to design novel antibiotics that target conserved DEAD-box domains in pathogenic organisms.