Central domain assembly: Thermodynamics and kinetics of S6 and S18 bindingto an S15-RNA complex

The 30 S ribosomal subunit assembles in vitro through the hierarchical bind ing 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 b y binding of S15 to 16 S rRNA, followed by the cooperative binding of S6 an d S18. The prior binding of S6 and S18 is required for binding of S11 and S 21. We have studied the mechanism of the cooperative binding of S6 and S18 to the S15-rRNA complex by isothermal titration calorimetry and gel mobilit y 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 degreesC. The S6:SIS heterod imer binds to the S15-rRNA complex with an equilibrium dissociation constan t of 2.7 nM at 40 degreesC. 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 aff inity of S6:S18 for the RNA by at least four orders of magnitude. The kinet ics of S6:S18 binding to the S15-rRNA complex are slow, with an apparent bi molecular rate constant of 8.0 x 10(4) M-1 s(-1) and an apparent unimolecul ar dissociation rate of 1.6 x 10(-4) s(-1). These results, which are consis tent 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 observ ed S15-mediated cooperative binding of S6 and S18 in the ordered assembly o f a multi-protein ribonucleoprotein complex. (C) 2001 Academic Press.