The objective of this proposal is to determine the structural basis of how the Stem-Loop Binding Protein (SLBP) functions to regulate histone mRNA processing and translation. Previous biochemical, and genetic studies have demonstrated that SLBP is the single most important trans-acting factor that plays an essential role in histone mRNA metabolism by forming a high affinity complex with a conserved stem-loop at the 3' end of replication-dependent histone mRNAs. The SLBP/RNA complex is important for the recruitment and assembly of multi-protein-RNA complexes that regulate pre-mRNA processing, translation, and degradation of histone mRNAs. Although a wealth of biochemical and genetic information exists for SLBP, the molecular basis for the SLBP-RNA interaction remains to be elucidated. We will take a multi-disciplinary approach to structurally characterize the RNA binding and processing domain (RPD) of SLBP, both free and bound to histone mRNA, using NMR and mass spectrometry. The experiments described in this proposal will complement ongoing functional studies in my laboratory, provide mechanistic information relating to SLBP function, and test currently proposed models as to how SLBP regulates histone metabolism in vivo.
The specific aims of the proposal are 1) to determine the structural and dynamic properties of the Drosophila SLBP RNA binding and processing domain (dSLBP RPD) in the absence of RNA using high resolution NMR spectroscopy and FT-ICR H/D exchange mass spectrometry, 2) to determine the structural and dynamic properties of the dSLBP RPD stem-loop histone mRNA complex using high resolution NMR spectroscopy and FT-ICR H/D exchange mass spectrometry, 3) to characterize the biological and biochemical properties of sSLBP RPD mutants impaired in RNA binding and RNA processing, 4) to structurally characterize dSLBP RPD mutants by NMR Spectroscopy, and 5) to provide a structural basis for sequence specific recognition of the translation initiation factors AD2 and elF4G by SLBP. These studies will provide new information on structure/function relationships for this biological important protein and also lay the groundwork for long-term goals which are (i) to understand the molecular determinants for assembly of multi-protein complexes at the 3' end of histone mRNAs and (ii) to develop RNA binding domains with novel specificities that can be used as biosensors or reagents for cell biological studies. ? ?
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