Binding of the protein S4 to 16S rRNA is critical for the subsequent binding of other ribosomal proteins, and mutations in S4 affect the accuracy of translation. S4 regulates its own translation and that of three other ribosomal proteins by binding to its own messenger RNA. In order to obtain an understanding of S4 function at the molecular level, we have solved the solution structure of S4delta41 by multidimensional heteronuclear NMR spectroscopy. Although S4 delta41 lacks the 41 N- terminal amino acids of intact S4, S4delta41 binds RNA targets almost as well as S4. The protein fold consists of two subdomains, with a concentration of positive charge along the crevice between the subdomains. The crevice may function as an RNA binding site, a hypothesis which is being checked by mutagenesis studies. The relative orientation of the two subdomains with respect to the long axis of the molecule was not precisely determined in the original NMR structure. This has been remedied by including dipolar couplings, measured in two different liquid crystalline media, in the list of NMR restraints. The refined solution structure is in close agreement with an independently determined X-ray structure, and this work is among the first demonstrations of the use of dipolar couplings to determine subdomain orientations of proteins in solution. We have recently obtained nearly complete signal assignments of intact S4, and preliminary analysis shows that the N-terminal 41 residues (absent from S4delta41) are highly flexible, and sample numerous conformations. NOESY evidence indicates that the structure of the remainder of the protein is very similar to that of S4delta41. Now that we have completed the assignments of intact S4 and characterized its structure, our goals are to (a) determine if the N-terminal 41 residues assume an ordered structure when S4 binds to RNA and (b) compare the residues of S4 and S4delta41 that are involved in binding to specific RNA targets. The first RNA target under study is a 112 nucleotide portion of the S4 mRNA molecule. This RNA has an unusual double pseudoknot secondary structure. Amino acids that interact with this target will be identified from chemical shift perturbations, induced by the RNA, in HSQC spectra of S4 and S4delta41.
| 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 |
