Binding of the protein S4 to 16S rRNA is critical for the subsequent binding of other ribosomal proteins, and regulates its own translation and that of three other ribosomal proteins. We solved the solution structure of its mRNA binding domain binding domain (S4delta41, 159 residues). S4delta41 has a novel fold with a positively charged cleft running along two distinct domains, suggesting a likely RNA-binding site. We examined the binding of S4delta41 to 112 nucleotides from the mRNA, by mapping changes chemical shifts onto the residues in the protein. Although the residues with perturbed shifts were on the positively charged face of the protein, broadening of certain peaks suggests that the protein-RNA complex is dynamic. Moreover, band shift assays showed that nonspecific binding occurs. We have probed pH, buffer, salt concentration, and temperature, but have not yet identified conditions which abolish non-specific binding. We have shown that the structure of the C-terminal 158 residues of the intact protein is the same that of S4delta41, while the 45 N-terminal residues are very flexible. Chemical shifts and NOESY restraints indicated that residues, S12RRL15 and P30YPP33, adopt transiently ordered structures. In addition, 15N relaxation data indicated that their motion is more restricted than that of other residues in the N-terminus. A BLAST search revealed that R13, R14, P33, G34, and H36 are completely conserved in S4 molecules from both eubacterial and chloroplast ribosomes. The modeled structures indicated that the two conserved arginine side chains of S12RRL15 extend out into solvent in a parallel fashion, suggesting a possible RNA-binding site, and that P30YPP33 form a nascent turn of a polyproline II helix, a protein-protein interaction motif. L11 is a large subunit protein that is essential for efficient protein synthesis. We previously determined the solution structure of it C-terminal domain and have now obtained spectra of intact L11. After finding conditions that maximize solubility we will determine its structure. L11 has not be located in the 2.4? crystal structure of large subunit of the ribosome. The difficulty of placing L11 may reflect L11's proposed role as a molecular switch. NMR is well-suited to determine if the linker between the N- and C-terminal domains is flexible, thereby allowing L11 to act as a molecular switch
| Sayers, E W; Gerstner, R B; Draper, D E et al. (2000) Structural preordering in the N-terminal region of ribosomal protein S4 revealed by heteronuclear NMR spectroscopy. Biochemistry 39:13602-13 |
| Markus, M A; Gerstner, R B; Draper, D E et al. (1999) Refining the overall structure and subdomain orientation of ribosomal protein S4 delta41 with dipolar couplings measured by NMR in uniaxial liquid crystalline phases. J Mol Biol 292:375-87 |
