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
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
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DOI：
10.1021/bi00407a002
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发表时间：
1988-04-05
期刊：
影响因子：
2.9
通讯作者：
BUJALOWSKI, W
中科院分区：
文献类型：
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作者：
LOHMAN, TM;BUJALOWSKI, W
我们通过监测固有蛋白荧光的猝灭，研究了寡核苷酸dT(pT)34与大肠杆菌SSB蛋白的结合与NaCl和MgCl2浓度（25℃，pH 8.1）的关系。我们发现每个单链结合（SSB）蛋白四聚体的dT(pT)34有两个结合位点，根据盐浓度的不同，每个位点具有不同的亲和力。在200 mM NaCl下，我们观察到dT(pT)34与SSB四聚体中的两个结合位点的结合几乎是化学计量的，尽管亲和性的差异仍然很明显。然而，当NaCl浓度降低时，dT(pT)34对SSB四聚体第二位点的整体亲和力显著降低。在1.5 mM NaCl下，即使dT(pT)34的摩尔量超过10倍，也只有一个dT(pT)34分子能与SSB四聚体结合。MgCl2在比NaCl低100倍的浓度下能有效促进dT(pT)34第二分子的结合。这种结合行为反映了SSB四聚体的固有特性，因为它也可以在较小的寡核苷酸结合上观察到，最简单的解释是SSB四聚体内的DNA结合位点之间存在盐依赖性负协同性。这种现象也导致了两种ssb -单链（ss）多核苷酸结合模式之间的转变，每个四聚体覆盖35和56个核苷酸[Bujalowski， W., and Lohman， T. M. (1986) Biochemistry 25, 7799-7802]。极端负协同性稳定了（SSB）35的结合模式，在这种模式下，SSB四聚体仅与SSB DNA的两个亚基紧密结合，而其他两个亚基保持不结合。在较高的盐浓度下，负协同性降低，结果是所有四个SSB亚基都可以与SSB DNA相互作用，如（SSB）56和（SSB）65结合模式。讨论了这种负合作可能的生物学意义。
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.degree.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., and 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. 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)65 binding modes. The possible biological significance of this negative cooperativity is discussed.