Interactions of bacteriophage T4-coded gene 32 protein with nucleic acids. II. Specificity of binding to DNA and RNA.

In this paper we examine the specificity of the cooperative binding (in the polynucleotide mode) of bacteriophage T4-coded gene 32 protein to synthetic and natural single-stranded ~iucleic acids differing in base composition and sugar type. I t is shown by competition experiments in a tight-binding (low salt) environment that there is a high degree of binding specificity under these (protein-limiting) conditions, with one type of nucleic acid lattice binding gene 32 protein to saturation before any binding to the competing lattice takes place; it is&lso shown that the same differential specificities apply a t high salt concentrations. Procedures developed in the preceding paper (Kowalczykowski et al., 1980) are used t o measure the net binding affinities (Kw) of gene 32 protein to a variety of polynucleotides, as well as to determine individual values of K and w for some systems. For all polynucleotides, virtually the entire specificity and salt dependence of binding of Kw appears to be in K. In-0.2 M-N ~ C ~ , the net binding affinities (Kw) range from-l o 6 to-10'' M-' ; in order of increasing affinities we find: poly(rC) <poly(rU) <poly(rA) <poly(dA) <poly(dC) <poly(dU) < poly(r1) <poly(dI) <poly-(dT). In general, Kw for a particular homopolyribonucleotide at constant salt concentration is 10' to lo4 smaller than Kw fhr the corresponding homopoly-deoxyribopolynucleotide. Values of Kw for randonily copolymerized poly-nucleotides and for natural DKA fall a t the compositionally weighted average of the values for the individual homopolynucleotides (except for poly(dT), which appears to bind somewhat tighter), indicating that the net affinity represents the sum of the binding free energy contributions of the individual nucleotides. I t is shown that these results, on a competition basis under physiological salt conditions, can account quantitatively for the autogenous regulation of the synthesis of gene addition, these results suggest possible mechanisms by which gene 32 messenger RNA might be specifically recognized (by gene 32 protein) and functionally discriminated from the other mRNAs of phage T4.