Structure-Function Studies of DEAD-Box RNA Chaperones

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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.