Comparative Analysis of RNA Helicases in E. coli

描述（由申请人提供）：DEAD-盒RNA解旋酶是一类多功能的保守蛋白，其特征在于它们能够解开RNA双链体，据信其在调节RNA功能中起重要作用。然而，许多死亡盒蛋白的研究很少，因此它们的细胞功能是未知的.没有这些知识，对RNA代谢的详细了解仍然是不完整的。我们建议研究两个E. coli DEAD盒RNA解旋酶SrmB和DeaD，它们参与RNA代谢的不同方面。我们的初步数据表明：（i）SrmB和DeaD各自促进非加工性核糖核酸外切酶，多核苷酸磷酸化酶（PNDase），以消化体内结构化RNA;（ii）缺乏任何一个因子导致8%的E.（iii）srmB和deaD菌株的冷敏感性生长表型可以通过过量表达核糖体RNA加工和修饰因子来抑制;（iv）E.大肠杆菌DEAD盒解旋酶刺激多聚腺苷酸聚合酶（PAP）的功能，PAP是一种使细胞转录物聚腺苷酸化并靶向其降解的酶。因此，这些蛋白质中的每一种在RNA周转中具有多种作用。使用遗传和生物化学方法的组合，拟议的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或DeaD刺激PNTR功能进行周转的细胞转录本。我们还将生化表征负责这些属性的RNA决定簇。其次，我们将确定直接与SrmB或DeaD在细胞中相互作用的转录本，并研究这些因子的差异转录调控机制。第三，我们将研究由于核糖体RNA加工因子的过表达而抑制srmB和deaD冷敏感性生长缺陷的基础。第四，我们将分析通过DEAD盒解旋酶刺激PAP的机制和后果。总的来说，这些研究应该提供深入了解目标选择的基础，与RNA加工酶的功能相互作用和两个重要的死亡盒蛋白的细胞功能。这些发现应该与尚未详细表征的死亡盒蛋白广泛相关。项目叙述 通过提供对RNA代谢的更深入了解，拟议的对DEAD盒RNA解旋酶的研究将为由于RNA功能缺陷引起的疾病提供更好的见解。这种RNA解旋酶的先进知识也有助于设计针对病原生物中保守的DEAD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.