Structure-Function Studies of DEAD-Box RNA Chaperones

大卫·麦凯MCB-98-745281。技术“DEAD-box”RNA解旋酶结构域是一个保守的~400个残基的蛋白质模块，在RNA代谢的广泛活动中发挥重要作用，包括核糖体组装、启动多肽翻译、mRNA剪接、RNA降解等。Helicasedomain被认为促进了特定RNA结构的重排，这些结构不是精确控制的，依赖于ATP的反应。DEAD-BOX结构域家族的不同代表作为单独的“独立”蛋白，在长度长达~1300个残基的更大的多结构域多肽的背景下作为亚域，也作为多蛋白复合体的亚单位。本研究的总体目标是确定DEAD-BOX蛋白的结构和RNA结合特异性。首先，一种具有代表性的DEAD-box蛋白的X射线晶体结构，酵母中的延伸因子eIF4A。都会得到解决。这将提供解旋酶模块的原型结构。将该结构纳入后续实验的设计中，(I)将表达和纯化几个在生物学功能上有显著差异的真细菌死盒蛋白，(Ii)将使用体外选择方法确定它们的RNA结合特异性。使用从选择实验中产生的一致的RNA靶序列，将合成定义的RNA寡核苷酸，并将直接测量它们与其同源死盒蛋白的相互作用。此外，还将尝试RNA-蛋白质复合体的结晶，以解决其结构问题。非技术性的RNA代谢是细胞存活所必需的功能。一组不同的蛋白质参与信使RNA的剪接和降解，通过与信使RNA的特定相互作用启动多肽翻译，以及在核糖体中组装RNA-蛋白质复合体。虽然这些活动中的每一个都利用了一组独特的蛋白质，但它们共享一个共同的“RNA伴侣”功能，该功能是由一个高度保守的RNA解旋酶结构域执行的。这个结构域的大小约为400个氨基酸，必须进行对这些蛋白质参与的整个生物活性光谱至关重要的非化学反应。这项工作的目标是了解这个蛋白质模块在RNA代谢的生化机制中的参与。实现这一点的方法将是首先解决这个RNA伴侣结构域的三维结构，然后确定其不同生物学功能的特异性基础。第一个目标将使用X射线结晶学技术来实现，第二个目标将使用体外选择技术来确定蛋白质结合的特定RNA序列，并使用结晶学来解决蛋白质-RNA复合体的结构。

David McKayMCB-98-745281. TechnicalThe "DEAD-box" RNA helicase domain is a conserved ~400 residue proteinmodule that has an essential role in a broad spectrum of activities in RNAmetabolism, including ribosome assembly, initiation of polypeptidetranslation, mRNA splicing, RNA degradation, and others. The helicasedomain is thought to facilitate rearrangments of specific RNA structures inprecisely controlled, ATP-dependent reactions. Different representatives ofthe DEAD-box domain family function as individual "stand alone" proteins,as subdomains in the context of larger, multidomain polypeptides up to~1300 residues in length, and also as subunits of multiprotein complexes.The overall goal of this study is to determine the structures and RNAbinding specificities of DEAD-box proteins. First, the x-raycrystallographic structure of a representative DEAD-box protein, theelongation factor eIF4a from yeast. will be solved. This will provide a prototype structure of the helicase module. Incorporating the structure into the design of subsequent experiments, (i) several eubacterial DEAD-box proteins which differ significantly in biological function willbe expressed and purified, and (ii) their RNA binding specificities will bedetermined using in vitro selection methods. Using the consensus RNA targetsequences that emerge from the selection experiments, defined RNA oligonucleotides will be synthesized and their interaction with their cognate DEAD-box proteins will be measured directly. Additionally,crystallization of RNA-protein complexes will be attempted with the goal ofsolving their structure.2. Non-technicalRNA metabolism is a required function for cell viability. A diverse set ofproteins participate in activities such as splicing and degradation ofmessenger RNA, initiation of polypeptide translation through specificinteractions with messenger RNA, and assembly of the RNA-proteincomplexes in the ribosome. Although each of these activities utilizes aunique set of proteins, they share a common "RNA chaperone" functionthat is carried out by a highly conserved RNA helicase domain. Thisdomain, which is about 400 amino acids in size, must carry out abiochemical activity that is essential to the entire spectrum of thebiological activities in which these proteins participate. The goal ofthis work is to understand the participation of this protein module in thebiochemical mechanisms of RNA metabolism. The method for accomplishingthis will be to first solve the three-dimensional structure of this RNAchaperone domain, and then to determine the basis of its specificity indifferent biological functions. The first goal will be accomplished usingthe technique of x-ray crystallography, and the second will use both invitro selection techniques to determine specific RNA sequences to which theproteins bind, and crystallography to solve the structures of protein-RNAcomplexes.