Comparative Analysis of RNA Helicases in E. coli

描述（由申请人提供）：DEAD-box RNA解旋酶是一类多用途的保守蛋白，其特征是它们具有解开RNA双链的能力，被认为在调节RNA功能中起重要作用。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。我们拟研究两种大肠杆菌DeaD -box 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) smp和DeaD各自促进非过程性外核糖核酸酶多核苷酸磷酸化酶（PNPase）在体内消化结构化RNA；（ii）任何一个因子的缺失导致8%的大肠杆菌转录物的差异调控，可能是通过对RNA构象和周转的影响；（iii）过表达核糖体RNA加工修饰因子可抑制srmB和deaD菌株的冷敏生长表型；（iv）大肠杆菌DEAD-box解旋酶刺激聚(A)聚合酶（PAP）的功能，PAP是一种聚腺苷化细胞转录物并将其降解的酶。因此，这些蛋白质中的每一种在RNA转换中都有多种作用。利用遗传和生化方法的结合，提出的研究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smp或DeaD刺激PNPase功能进行转换的细胞转录本。我们也将生化表征负责这些特性的RNA决定因子。其次，我们将识别细胞中直接与smp或DeaD相互作用的转录本，并研究这些因子对转录本差异调控的机制。第三，我们将研究核糖体RNA加工因子过度表达抑制smrmb和deaD冷敏感生长缺陷的基础。第四，我们将分析DEAD-box解旋酶刺激PAP的机制和后果。总的来说，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种重要DEAD-box蛋白的细胞功能提供见解。这些发现应该与尚未被详细描述的DEAD-box蛋白广泛相关。项目的叙述

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.