Central domain assembly: thermodynamics and kinetics of S6 and S18 binding to an S15-RNA complex.
The 30 S ribosomal subunit assembles in vitro through the hierarchical binding of 21 ribosomal proteins to 16 S rRNA. The central domain of 16 S rRNA becomes the platform of the 30 S subunit upon binding of ribosomal proteins S6, S8, S11, S15, S18 and S21. The assembly of the platform is nucleated by binding of S15 to 16 S rRNA, followed by the cooperative binding of S6 and S18. The prior binding of S6 and S18 is required for binding of S11 and S21. We have studied the mechanism of the cooperative binding of S6 and S18 to the S15-rRNA complex by isothermal titration calorimetry and gel mobility shift assays with rRNA and proteins from the hyperthermophilic bacterium Aquifex aeolicus. S6 and S18 form a stable heterodimer in solution with an apparent dissociation constant of 8.7 nM at 40 degrees C. The S6:S18 heterodimer binds to the S15-rRNA complex with an equilibrium dissociation constant of 2.7 nM at 40 degrees C. Consistent with previous studies using rRNA and proteins from Escherichia coli, we observed no binding of S6 or S18 in the absence of the other protein or S15. The presence of S15 increases the affinity of S6:S18 for the RNA by at least four orders of magnitude. The kinetics of S6:S18 binding to the S15-rRNA complex are slow, with an apparent bimolecular rate constant of 8.0 x 10(4) M(-1) s(-1) and an apparent unimolecular dissociation rate of 1.6 x 10(-4) s(-1). These results, which are consistent with a model in which S6 and S18 bind as a heterodimer to the S15-rRNA complex, provide a mechanistic framework to describe the previously observed S15-mediated cooperative binding of S6 and S18 in the ordered assembly of a multi-protein ribonucleoprotein complex.
Recht
M I
MI
Department of Molecular Biology, MB33 and the Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Rd., La Jolla, CA 92037, USA.
Williamson
J R
JR
D001419:Bacteria / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D001483:Base Sequence; D002151:Calorimetry; D019281:Dimerization; D029968:Escherichia coli Proteins / Q000378:metabolism; D007700:Kinetics; D046911:Macromolecular Substances; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021122:Protein Subunits; D012336:RNA, Ribosomal, 16S / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D038601:Ribosomal Protein S6; D012269:Ribosomal Proteins / Q000737:chemistry / Q000378:metabolism*; D012270:Ribosomes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013816:Thermodynamics
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D046911:Macromolecular Substances; D021122:Protein Subunits; D012336:RNA, Ribosomal, 16S; D038601:Ribosomal Protein S6; D012269:Ribosomal Proteins; C036004:ribosomal protein S15; C041184:ribosomal protein S18