This proposal outlines a plan to investigate RNA-protein interactions that regulate mRNA translation. Three different systems will be investigated using a biochemical approach to further our understanding of important regulatory complexes that have been identified genetically. The first system to be studied is the complex between the Caenhorhabditis elegans GLD-1 protein and the TGERNA site from the mRNA of tra-2. The GLD-1:TGE complex is involved in translational regulation oftra-2, in the control of germline development. GLD-1 is a member of the STAR class of proteins, which are characterized by a KH (hnRNP K homology) domain, flanked by a quaking-1 andquaking-2 domain. The STAR domain is an elaborated version of the KH RNA binding domain. The second system to be studied is a multicomponent RNA-protein complex involved in localization and translational regulation of the Saccharomyces cerevesiae ASH1 mRNA. ASH1 is localized to the tip of a budding daughter cell, and the ASH1 protein regulates mating type switching. A number of proteins are associated with RNA signals in the ASH1 mRNA including, She2, She3, and Khd1. Khd1 is a protein with 3 simple KH domains, and it binds to the same RNA structural element implicated in binding of She2p, which contains no known RNA binding sequence motifs. The third system to be studied is the ribosomal protein S15 from Bacillus stearothermophilus bound to a three-helix junction that is a translational regulatory signal in the mRNA for the S15. S15 autoregulates its own translation by binding to this element in the 5'-untranslated region. The mRNA site is distinct from the three-helix junction found in the 16S rRNA, and we will make a comparison of how two different RNAs are recognized by the same protein.
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