We have extended our investigations of the multiple binding modes that form between the Escherichia coli single strand binding (SSB) protein and single-stranded DNA (Lohman, T.M. & Overman, L.B. (1985) J. Biol. Chem. 260, 3594-3603; Bujalowski, W & Lohman, T.M. (1986) Biochemistry 25, 7799-7802) by examining the effects of anions, pH, BaCl2, and protein binding density on the transitions among these binding modes. 'Reverse' titrations that monitor the quenching of the intrinsic tryptophan fluorescence of the SSB protein upon addition of poly(dT) have been used to measure the apparent site size of the complex at 25°C in pH 8.1 and 6.9 as a function of NaF, NaCl, NaBr, and MgCl2 concentrations. Under all conditions in which 'reverse' titrations were performed, we observe three distinct binding modes with site sizes of 35 ± 2, 56 ± 3, and 65 ± 3 nucleotides/SSB tetramer; however, the transitions among the three binding modes are strongly dependent upon both the cation and anion valence, type, and concentration as well as the pH. A net uptake of both cations and anions accompanies the transition from the (SSB)35 to the (SSB)56 binding mode at pH 6.9, whereas at pH 8.1 this transition is anion-independent, and only a net uptake of cations occurs. The transition from the (SSB)56 to the (SSB)65 binding mode is dependent upon both cations and anions at both pH 6.9 and 8.1 (25°C), and a net uptake of both cations and anions accompanies this transition. We have also examined the transitions by monitoring the change in the sedimentation coefficient of the SSB protein-poly(dT) complex as a function of MgCl2 concentration (20°C, pH 8.1) and observe an increase in S(20,10), which coincides with the increase in apparent site size of the complex, as measured by fluorescence titrations. The frictional coefficient of the complex decreases by a factor of two in progressing from the (SBB)35 to the (SSB)65 binding mode, indicating a progressive compaction of the complex throughout the transition. The transition between the (SBB)35 and the (SSB)56 complex is dependent on the protein binding density, with the lower site size (SSB)35 complex favored at higher binding density. These results indicate that the transitions among the various SSB protein-single-stranded DNA binding modes are complex processes that depend on a number of solution variables that are thermodynamically linked. Thus, caution must be exercised when comparing data collected under different sets of solution conditions in any experiments involving the E. coli SSB protein in vitro.
|Number of pages||12|
|Journal||Journal of Biological Chemistry|
|State||Published - 1988|