Negative cooperativity within individual tetramers of Escherichia coli single strand binding protein is responsible for the transition between the (SSB)35 and (SSB)56 DNA binding modes.
We have examined the binding of the oligonucleotide dT (pT)34 to the Escherichia coli SSB protein as a function of NaCl and MgCl2 concentration (25 degrees C, pH 8.1) by monitoring the quenching of the intrinsic protein fluorescence. We find two binding sites for dT(pT)34 per single strand binding (SSB) protein tetramer, with each site possessing widely different affinities depending on the salt concentration. At 200 mM NaCl, we observe nearly stoichiometric binding of dT(pT)34 to both binding sites within the SSB tetramer, although a difference in the affinities is still apparent. However, when the NaCl concentration is lowered, the overall affinity of dT(pT)34 for the second site on the SSB tetramer decreases dramatically. At 1.5 mM NaCl, only a single molecule of dT(pT)34 can bind per SSB tetramer, even with a 10-fold molar excess of dT(pT)34. MgCl2 is effective at 100-fold lower concentrations than NaCl in promoting the binding of the second molecule of dT(pT)34. This binding behavior reflects an intrinsic property of the SSb tetramer, since it is also observed upon binding of smaller oligonucleotides, and the simplest explanation is that a salt-dependent negative cooperativity exists between DNA binding sites within the SSB tetramer. This phenomenon is also responsible for the transition between the two SSB-single strand (ss) polynucleotide binding modes that cover 35 and 56 nucleotides per tetramer [Bujalowski, W., & Lohman, T. M. (1986) Biochemistry 25, 7799-7802]. Extreme negative cooperativity stabilizes the (SSB)35 binding mode, in which the SSB tetramer binds tightly to ss DNA with only two of its subunits while the other two subunits remain unligated.(ABSTRACT TRUNCATED AT 250 WORDS)
Lohman
T M
TM
Department of Biochemistry and Biophysics, Texas A & M University, College Station 77843.
Bujalowski
W
W
D001665:Binding Sites; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism*; D007700:Kinetics; D046911:Macromolecular Substances; D008274:Magnesium / Q000494:pharmacology; D015636:Magnesium Chloride; D009838:Oligodeoxyribonucleotides / Q000378:metabolism; D009994:Osmolar Concentration; D011485:Protein Binding; D012965:Sodium Chloride / Q000494:pharmacology
D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides; C027903:oligo (dT); D015636:Magnesium Chloride; D012965:Sodium Chloride; D008274:Magnesium