This proposal outlines a broadly based investigation into RNA- protein recognition. Combining biochemical approaches and NMR structural studies, we will investigate the nature of specific interactions between RNAs and proteins. The central themes of this proposal will be RNA-mediated recognition of alpha helices and protein-mediated recognition of RNA internal loops. Structural studies will reveal the detailed nature of the intermolecular interface, while biochemical studies provide the key to determine which interactions at the interface are thermodynamically important for providing affinity and specificity.
The Specific Aims are: 1) Determine the structure of Rev-RRE complexes with altered specificity. The Rev N40Q mutant was selected as a suppressor of the A73G mutation in the RRE RNA and the Rev Q36G mutation is a suppressor of the G47A mutation in the RRE. We will determine the structure of these two double mutant complexes to determine how these mutations provide mutual compensation in the context of the remainder of the binding site. 2) Biochemical and structural studies of the phi21 N(8-29)-boxB RNA complex. The N protein is a phage transcriptional antitermination protein that contains an amino-terminal basic region that mediates recognition to boxB of the nut site RNA target. We have identified suitable peptides from the lambdoid phage phi21 that bind to the phi21 boxB RNA using the PACE assay developed in our laboratory. We plan to determine the structure of this complex using NMR spectroscopy. The structural studies will be followed by mutagenesis studies of the RNA and peptide to assess the thermodynamic contributions of residues at the intermolecular interface to affinity and specificity of binding. 3) Biochemical and structural studies of the yeast ribosomal protein L30 complex. The yeast S. cerevisiae L30 protein (formerly L32) autoregulates its own expression at the level of splicing and translation by binding to an internal loop sequence at the 5' end of its own mRNA. We have nearly completed the structure determination of this RNA-protein complex by NMR, revealing a novel interaction between three loops on the protein and three nucleotides in the RNA internal loop. We plan to refine the structure to completion, and continue NMR and biochemical studies to evaluate the contribution of the amino acids at the RNA-protein interface to the specificity and affinity of RNA binding.
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