Sequence Specific Interaction of R17 Coat Protein With Its Rna Binding Site
The interaction between RNA bacteriophage R17 coat protein and its RNA binding site for translational repression was studied as an example of a sequence-specific RNA-protein interaction. The binding site consists of fewer than 20 contiguous nucleotides which form a small hairpin. The thermodynamic validity of a simple nitrocellulose filter assay for complex formation is established. This assay is used to show that the binding between the coat protein and a synthetic 21 nucleotide RNA hairpin conforms to a simple bimolecular equilibrium reaction. Unit stoichiometry and a dissociation constant of (TURN)1 nM are obtained at 2(DEGREES)C in a buffer containing 0.19 M salt. The interaction is shown to be highly specific since a variety of RNAs failed to compete with the 21 nucleotide fragment for coat protein binding. The kinetics of the reaction are shown to be consistent with the equilibrium constant and indicate a diffusion-controlled reaction. The temperature dependence of the equilibrium constant gives (DELTA)H = -19 kcal/mole, which is partially offset by a (DELTA)S = -29 cal/mole-deg to give a (DELTA)G of -11 kcal/mole at 2(DEGREES)C. The binding reaction has a pH optimum centered at pH 8.5, but pH has no effect on (DELTA)H. While the interaction is insensitive to the type of monovalent cation, the affinity decreases with the lyotropic series among monovalent anions. The ionic strength dependence of the equilibrium constant indicates that ionic contacts contribute to the interaction, but most of the binding free energy arises from nonelectrostatic interactions. Twenty-three variants of the binding fragment were synthesized enzymatically and their coat protein affinities determined. The minimum binding site size deduced from truncated variants is eighteen nucleotides. Individual nucleotide substitutions in the hairpin loop reveal unexpectedly large decreases in affinity, suggesting that each of these single stranded residues may be the site of more than one contact with the protein. Although the secondary structure of the binding site appears to be required for binding, specific contact sites in helical regions could not be distinguished from the need to maintain the correct orientation of contacts in unstructured regions. Similarly, a bulged adenosine is essential for binding, but its role as a point of contact could not be established. A total of five sites of contact between the protein and the RNA are identified. These lie along one face of the helical RNA and span a distance compatible with the size of the coat protein calculated from its known sedimentation constant.
University of Illinois - Urbana-Champaign
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University of Illinois at Urbana-Champaign