A fluorescence study of the binding of poly(1, N6-ethenoadenylic acid) to Escherichia coli initiation Factor 3

Abstract

The binding of initiation Factor 3 (IF3) to poly(1, N 6 -ethenoadenylic acid) [poly(ϵA)] was investigated by fluorescence spectroscopy. At low salt concentrations, IF3 evokes an increase in the fluorescence intensity of poly(ϵA) due to the unstacking of the nucleotide bases. The poly(ϵA) fluorescence enhancement titrates to an endpoint of 13 ± 2 nucleotide residues per IF3. The maximum poly(ϵA) fluorescence enhancement, at lattice saturation, decreases with increasing salt concentration. Even though IF3 does not produce a large fluorescence increase between 75 and 200 m m NaCl concentration, the protein still binds to poly(ϵA) at these salt concentrations as measured by sedimentation partition chromatography; the value of K obs for the IF3-poly(ϵA) interaction is comparable to that of other synthetic polynucleotides. The binding of IF3 to poly(A) at 150 and 200 m m NaCl induces an increase in nucleotide base-base separation as determined by CD, yet IF3-induced disruption of base stacking of poly(ϵA) at these same salt concentrations is not detected by fluorescence. It is likely that IF3 binds primarily to the phosphate backbone of poly(ϵA) at low salt concentrations, producing an increase in the fluorescence intensity. But, at higher salt concentrations, the aromatic amino acids intercalate between the nucleotide bases quenching the poly(ϵA) fluorescence.