RIBOSOMAL PROTEIN S4 INTERACTIONS WITH MRNA AND RRNA

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Complexes of RNA and protein (RNPs) carry out many essential functions in the cell, including translation. How proteins within these complexes recognized specific sites, promote RNA folding, and contribute to function are important questions. The goal of this project is to explore these question for a ribosomal protein, S4, that has dual functions: it is a key protein in ribosome assembly, binding and organizing a approximately 500 nucleotide domain of the small subunit RNA, and also binds a 110 nucleotide pseudoknot structure in the alpha mRNA, where it represses translation by a novel "entrapment" mechanism. The protein is organized into at least three regions: an N-terminal segment (42 residues) is not required for ribosome assembly: a middle domain is highly conserved and mostly responsible for RNA recognition; a C-terminal domain may contribute to both RNA and protein interactions. The structure of a fragment containing the middle and C-terminal regions (159 residues), derived from Bacillus stearothermophilus S4, will be determined by NMR methods. Site- directed mutagenesis, selection approaches, and thermodynamic and kinetic measurements will be used to explore the following aspects of S4-RNA interactions: Determination of conserved surface residues involved in mRNA or rRNA binding; Comparison of the ways in which S4 binding is linked to folding of the ribosomal and messenger RNA recognition domains, paying particular attention to the role of Mg/2+ in promoting RNA folding and protein binding; Resolution of the translational repression mechanism by studies of S4 and 30S subunit interactions with the mRNA ribosome binding site; Studies of the cooperatively between S4 and other ribosomal proteins (S20 and S16) assembling with the 16S rRNA 5' domain. Results from these studies should contribute to a basic understanding of protein-RNA recognition mechanisms and RNA folding, as well as elucidation of specific S4 roles in translational regulation and ribosome assembly.