Modular Structure and Function of RNase lll, a dsRNA-Binding Protein

SimonsRNA结合蛋白参与基因表达的所有方面，包括复制、转录、翻译、核糖体和信使RNA加工和衰变、RNA剪接和编辑以及RNA运输和输出。 核糖核酸酶是这些事件中的关键参与者。 核糖核酸酶III（RNase III）是一种广泛保守的双链RNA（dsRNA）特异性内切酶，在RNA加工和降解中发挥重要或必不可少的作用。 每个RNase III单体（功能性二聚体的）含有dsRNA结合基序（dsRBM）的单个拷贝，该基序存在于大量已知或被认为结合dsRNA的不同蛋白质中。 RNase III还含有一个高度保守的催化结构域（catD），迄今为止这种酶是独一无二的。 最近的重要证据揭示了dsRBM如何作为独立折叠的蛋白质模块与dsRNA结合。 其他最近的证据表明，catD也是一个独立的模块，在特定条件下具有强大和特异性的内切核糖核酸酶活性。 这些研究表明，RNA酶III的dsRBM和catD亚结构域可能通过紧密接触网络相互作用，以稳定结合并产生更有效的催化位点。 其他模块蛋白质（包括其他RNA结合蛋白）内的亚结构域接触尚未得到很好的理解。 该研究项目包括通过明确的具体目标，在子域水平上对RNase III结构和功能进行综合遗传攻击。 关键的生物化学工作将直接测试遗传预测。 RNase III二聚化的主要决定因素将在体内检查，通过分离点突变在一个新的遗传筛选。 将仔细评估dsRNA底物结合二聚体稳定性的潜在贡献，并通过现有的充分表征的阻止结合的dsRBM点突变进行辅助。 还将寻找catD中的底物结合决定簇。 dsRBM和catD之间的潜在相互作用将进行仔细研究，与一个新开发的体内试验，其中RNase III活性是从单独表达的子域，以及与最近开发的原核双杂交系统和其他手段重建。 这些努力将产生重要的见解dsRNA结合，RNase III催化，和模块蛋白质的结构和功能一般。SimonsRNA是所有细胞中遗传物质的仓库之一。 RNA结合蛋白参与RNA功能的各个方面。 RNase III是一种这样的RNA结合蛋白。 它存在于大多数细胞中。 RNase III含有一个RNA结合模块（RBM），存在于大量结合RNA的相关蛋白质中。 RNase III还含有一个催化模块（catD），这是该蛋白质家族所特有的。 最近的重要证据表明，成果管理制和catD模块通过密切联系网络相互作用。 一般来说，模块化蛋白质中的亚结构域接触还没有得到很好的理解。 该研究项目将在模块化水平上对RNase III结构和功能进行综合遗传和生化攻击。 这些努力将产生重要的见解RNA结合，RNase III功能，模块化蛋白质的结构和功能一般。

SimonsRNA-binding proteins are involved in all aspects of gene expression, including replication, transcription, translation, ribosomal and messenger RNA processing and decay, RNA splicing and editing, and RNA transport and export. Ribonucleases are key players in these events. Ribonuclease III (RNase III) is a double-stranded RNA (dsRNA)-specific endonuclease that is widely conserved, playing vital or essential roles in RNA processing and decay. Each RNase III monomer (of the functioning dimer) contains a single copy of the dsRNA-binding motif (dsRBM) found in a large number of diverse proteins known or thought to bind dsRNA. RNase III also contains a highly conserved catalytic domain (catD), so far unique to this enzyme. Recent and important evidence reveals how the dsRBM binds to dsRNA as an independently folded protein module. Other recent evidence shows that catD is also an independent module, possessing robust and specific endoribonuclease activity under specific conditions. These studies suggest that the dsRBM and catD subdomains of RNase III likely interact with one another through a network of intimate contacts, to stabilize binding and to create a more efficient catalytic site. Subdomain contacts within otherwise modular proteins, including other RNA-binding proteins, are not well-understood. This research project comprises an integrated genetic assault on RNase III structure and function at the subdomain level, through well-defined specific aims. Key biochemical work will directly test genetic predictions. The principal determinants of RNase III dimerization will be examined in vivo, through the isolation of point mutations in a novel genetic screen. The potential contribution of dsRNA substrate binding to dimer stability will be carefully assessed, aided by existing well-characterized dsRBM point mutations that prevent binding. Substrate-binding determinants in catD will also be sought. Potential interactions between dsRBM and catD will be studied carefully, with a newly developed in vivo assay in which RNase III activity is reconstituted from the individually expressed subdomains, as well as with the recently developed prokaryotic two-hybrid system and other means. These efforts will yield important insights into dsRNA-binding, RNase III catalysis, and modular protein structure and function in general.SimonsRNA is one of the storehouses of genetic material in all cells. RNA-binding proteins are involved in all aspects of RNA function. RNase III is one such RNA-binding protein. It is found in most cells. RNase III contains an RNA-binding module (RBM) found in a large number of related proteins that bind RNA. RNase III also contains a catalytic module (catD), unique to this protein family. Recent and important evidence suggests that the RBM and catD modules interact with one another through a network of intimate contacts. In general, subdomain contacts in modular proteins are not well-understood. This research project will mount an integrated genetic and biochemical assault on RNase III structure and function, at the modular level. These efforts will yield important insights into RNA-binding, RNase III function, and modular protein structure and function in general.