Equilibrium binding of Escherichia coli single-strand binding protein to single-stranded nucleic acids in the (SSB)65 binding mode. Cation and anion effects and polynucleotide specificity.
The Escherichia coli single-strand binding (SSB) protein binds single-stranded (ss) nucleic acids in at least four distinct binding modes depending on the salt conditions [Lohman, T. M., & Overman, L. B. (1985) J. Biol. Chem. 260, 3594; Bujalowski, W., & Lohman, T. M. (1986) Biochemistry 25, 7799]. Equilibrium binding constants for the interaction of the E. coli SSB protein with poly(A), poly(U), poly(dA), and poly(dT) have been measured over a range of monovalent salt concentrations and types under conditions which favor only the high site size, (SSB)65 binding mode, which covers 65 nucleotides per SSB tetramer. The binding isotherms are analyzed by using a statistical thermodynamic model ("tetramer/octamer" model) that assumes cooperative binding of SSB is limited to the formation of octamers [Bujalowski, W., & Lohman, T. M. (1987) J. Mol. Biol. 195, 897] rather than the indefinite clustering of tetramers. The dependence of the intrinsic association equilibrium constant, Kobsd, and cooperativity parameter, omegoT/O, on salt concentration has been determined by titrations which monitor the fluorescence quenching of the SSB protein upon complex formation. In the (SSB)65 binding mode, SSB binds with only moderate cooperativity to ss nucleic acids [Lohman, T. M., Overman, L. B., & Datta, S. (1986) J. Mol. Biol. 187, 603]. The cooperativity parameter derived from the tetramer/octamer model, which represents the equilibrium constant for formation of a nucleic acid bound SSB octamer from two nucleic acid bound tetramers, has a value of omegaT/O = 410 +/- 120 and is independent of salt concentration and type for poly(dA), poly(U), and poly(A) (25 degrees C, pH 8.1). However, Kobsd decreases steeply with increasing salt concentration, such that alpha log Kobsd/alpha log [NaCl] = -7.4 +/- 0.5 for poly(U), -6.1 +/- 0.6 for poly(dA), and -6.2 +/- 0.3 for poly(A) (25.0 degrees C, pH 8.1). The SSB-poly(dT) affinity is too high to measure in buffers containing even 5 M NaCl; however, in 1.8-2.5 M NaBr, we measure alpha log Kobsd/alpha log [NaBr] = -5.7 +/- 0.7, with a lower value of omega T/O = 130 +/- 70. The polynucleotide specificity of the (SSB)65 binding mode (0.20 M NaCl, 25.0 degrees C, pH 8.1) is Kobsd(dT) greater than Kobsd(dC) much greater than Kobsd(ss M13 DNA) greater than Kobsd(I) greater than Kobsd(U) = 8Kobsd(dA) = 87Kobsd(A) much greater than Kobsd(C).(ABSTRACT TRUNCATED AT 400 WORDS)
Overman
L B
LB
Department of Biochemistry and Biophysics, Texas A&M University, College Station 77843.
Bujalowski
W
W
Lohman
T M
TM
D000838:Anions; D002412:Cations; D003198:Computer Simulation; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism*; D007700:Kinetics; D009994:Osmolar Concentration; D011072:Poly U / Q000378:metabolism; D011119:Polynucleotides / Q000378:metabolism; D011485:Protein Binding; D013379:Substrate Specificity
D000838:Anions; D002412:Cations; D004268:DNA-Binding Proteins; D011119:Polynucleotides; D011072:Poly U