Competitive and Cooperative Interactions Mediate RNA Transfer from Herpesvirus Saimiri ORF57 to the Mammalian Export Adaptor ALYREF
Figure 6
Fluorescent studies and simulations of the ternary complex formation between ORF57, O, fragment of viral RNA, R, and ALYREF, A.
(A) Simultaneous non-linear fit of normalized fluorescence-derived parameters ΔλNbcm (blue shift of emission signal) and ΔIN (fluorescence quenching) to the non-redundant three-equation model using DynaFit software, to obtain Kd for the ternary complex. The experimental values of thus determined KdOA+R, as well as Kd's for other complexes measured earlier, are summarized on two illustrative thermodynamic cycles for the ternary complex assembly presented on panels (B) and (C). Simulations (using COPASI software [67]) for these two possible cycles illustrate an increase in the concentration of ternary ORF57-RNA-ALYREF complex when ORF57 is added to 10 µM equimolar mixture of ALYREF and RNA, assuming the simplest four-state equilibrium model (B), or six-state model which additionally takes into account weak nonspecific ALYREF-RNA binding (C). The arrow marks a point where all the components of ternary complex are present in equimolar amounts. The presence of equimolar ORF57 significantly increases the concentration of RNA in complex with ALYREF (i.e., [OAR] vs [AR][O] = 0). The baseline concentrations [AR][O] = 0 are indicated on the panel (C) on the left, assuming two different conservative estimates for values of Kd for nonspecific ALYREF-RNA binding.