Negative Cooperativity within Individual Tetramers of Escherichia coil Single Strand Binding Protein Is Responsible for the Transition between the (SSB)35 and (SSB)56DNA Binding Modes

Timothy M. Lohman, Wlodzimierz Bujalowski

Research output: Contribution to journalArticle

64 Scopus citations

Abstract

We have examined the binding of the oligonucleotide dT(pT)34to the Escherichia coli SSB protein as a function of NaCl and MgCl2concentration (25 °C, pH 8.1) by monitoring the quenching of the intrinsic protein fluorescence. We find two binding sites for dT(pT)34per 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)34to 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)34for the second site on the SSB tetramer decreases dramatically. At 1.5 mM NaCl, only a single molecule of dT(pT)34can bind per SSB tetramer, even with a 10-fold molar excess of dT(pT)34. MgCl2is 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)35binding mode, in which the SSB tetramer binds tightly to ss DNA with only two of its subunits while the other two subunits remain unligated. At higher salt concentrations, negative cooperativity is reduced with the result that all four SSB subunits can interact with ss DNA, as in the (SSB)56 and (SSB)65binding modes. The possible biological significance of this negative cooperativity is discussed.

Original languageEnglish (US)
Pages (from-to)2260-2265
Number of pages6
JournalBiochemistry
Volume27
Issue number7
DOIs
StatePublished - Apr 1 1988
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry

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