The overall goal of this project is to understand the assembly of the central domain of the 30 S ribosomal subunit. This includes biochemical characterization of central domain protein binding to 16 S rRNA and structural characterization of ribonucleoprotein complexes that correspond to intermediates in ribosomal subunit assembly. The investigators begin by characterizing the complex of the primary binding protein S15 with a minimal binding site that is a 3-way junction RNA. In addition, the investigators will characterize S15 binding to a heterologous mRNA binding site. The studies of the S15-RNA complex serve as the basis for subsequent characterization of a series of ribonucleoprotein complexes that include additional proteins, S6, S18, S11 and S21. The investigators hope to reveal the specific mechanism of central domain assembly, as well as about general principles of RNP assembly.
The Specific Aims are: 1) Determine the structure of a 3-way junction subfragment of the S15 binding site. The investigators have identified a 40 nucleotide RNA that is a minimal 3-way junction fragment capable of undergoing a Mg++ -induced conformational change, but binds S15 with greatly reduced affinity. The investigators will determine the structure of the folded junction region using NMR spectroscopic methods. 2) Determine the structure of the minimal S15-rRNA complex. The investigators are taking a two-pronged approach to determining the structure of S15 (88 residues) with a 65 nucleotide minimal binding site, and they will apply both NMR and X-ray diffraction methodology to solve the structure. 3) Biochemical studies of the S15 mRNA interaction. S15 autoregulates its own translation by binding to an RNA structure in the 5'-untranslated region of S15 mRNA., but this structure is not obviously homologous to the rRNA binding site. The investigators will characterize the binding of the protein to the mRNA site to understand how this small protein can recognize two different structures. 4) Kinetic and thermodynamic studies of the S15 binding to rRNA. The investigators will extend their knowledge of S15 binding to the 3-way junction RNA by studying the kinetics of the conformational change that accompanies protein binding using time-resolved FRET and single molecule fluorescence methods. 5) Kinetic and thermodynamic studies of the 30 S central domain assembly. Immediately after the primary binding protein S15 binds to 16 s rRNA, S6 and S18 bind, followed by S11 and S21. The investigators will characterize an incremental series of RNA-protein complexes that correspond to assembly intermediates. 6) Structural studies of a series of assembly intermediates of the 30 S central domain. The investigators will structurally characterize the series of assembly intermediates identified in Aim $6 to complete the mechanistic picture of the assembling central domain.
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