A Model of PSI Dimerization: Destabilization of the C278−G303Stem−Loop by the Nucleocapsid Protein (NCp10) of MoMuLV

Abstract

We have shown that at low ionic strength (i.e., 100 mM NaCl) a short autocomplementary sequence spanning nucleotides C²⁸³ to G²⁹⁸ of MoMuLV RNA genome is involved in the process of PSI dimerization in vitro [Girard, P.-M., Bonnet-Mathonière, B., Muriaux, D., & Paoletti, J. (1995) Biochemistry 34, 9785−9794]. In order to identify other contributions of the PSI structure to RNA dimerization, we studied the kinetics of dimerization as a function of salt concentration of short RNA transcripts comprising or not the autocomplementary sequence C²⁸³−G²⁹⁸. We propose that, apart from the crucial role of this sequence in RNA dimerization, the 364−565 domain of PSI can interfere, in vitro, with the initiation of dimer formation. Intermolecular loop−loop recognitions involving the 364−565 domain could stabilize, in a salt concentration-dependent manner, a transient RNA dimer built around the loop−loop U²⁸⁸−A²⁹³ interaction. This dimer evolves toward a more stable structure which mainly corresponds to the annealing of two C²⁸³−G²⁹⁸ sequences. We also show that chemically synthesized NCp10 does not modify these steps but rather helps the system to pass over the energy barriers associated with the transition to stable RNA structures comprising the stem−loop C²⁷⁸−G³⁰³. Data obtained in the presence of NCp10 suggest a binding site size of 9 ± 1 nucleotides per protein at 37 °C and a 10−20-fold increase in the rate constant (i.e., k₁ = 24 000 ± 7000 M^-1 s^-1) of dimer formation.