Electrostatics in RNA - protein recognition

Proteins interact with the strong electrostatic potential of RNA generated by the negatively charged phosphate backbone. It is recognized that electrostatics play an essential role in protein-RNA recognition. The first part of this thesis uses parallel experimental measurements and theoretical calculations to investigate the energetics of electrostatic interactions in the complex formed between a 22 residue, α-helical peptide from the N protein of phage λ, and its cognate 19 nucleotide Box B RNA hairpin. Salt-dependent free energies were measured for both peptide folding from coil to helix and peptide binding to RNA. From these the salt-dependence of binding pre-folded, helical peptide to RNA was determined. The non-linear Poisson Boltzmann equation was used to calculate the same salt-dependence of the binding free energy and the results from the calculations are in excellent agreement with the measured values. Close agreement between experimental measurements and calculations was also obtained for two variant peptides in which either a basic or acidic residue was replaced with an uncharged residue, and for an RNA variant with a deletion of a single loop nucleotide. The calculations suggest that the strength of electrostatic interactions between a peptide residue and RNA varies considerably with environment, but that all 12 positive and negative N peptide charges contribute significantly to the electrostatic free energy of RNA binding, even at distances up to 11 Å from backbone phosphates. Calculations also show that the net release of ions that accompanies complex formation originates from rearrangements of both peptide and RNA ion atmospheres, and includes accumulation of ions in some regions of the complex as well as displacement of cations and anions from the ion atmospheres of the RNA and peptide, respectively. The second part of this thesis explores the role of electrostatics on the specificity between N peptide and box B RNA. To that end, two N peptide and two box RNA variants were designed, selectively destabilizing ionic and hydrogen bonding interactions, respectively. Circular dichroism and fluorescence experiments suggest that deletion of one charge gives rise to a non-specific complex. Similar effects were found when one hydrogen bond was destabilized. The results of this second part support the hypothesis that electrostatics provide a global complex stabilization and play an essential role in the specificity of the complex. In addition, experimental data suggest that folding transitions in the peptide and RNA enlarge the energy gap between specific and non-specific complexes.