ESCHERICHIA-COLI SINGLE-STRAND DEOXYRIBONUCLEIC-ACID BINDING-PROTEIN - STABILITY, SPECIFICITY, AND KINETICS OF COMPLEXES WITH OLIGONUCLEOTIDES AND DEOXYRIBONUCLEIC-ACID
ESCHERICHIA-COLI SINGLE-STRAND DEOXYRIBONUCLEIC-ACID BINDING-PROTEIN - STABILITY, SPECIFICITY, AND KINETICS OF COMPLEXES WITH OLIGONUCLEOTIDES AND DEOXYRIBONUCLEIC-ACID
复制标题
DOI：
10.1021/bi00521a040
复制
发表时间：
1981-01-01
期刊：
影响因子：
2.9
通讯作者：
MAASS, G
中科院分区：
文献类型：
--
作者：
KRAUSS, G;SINDERMANN, H;MAASS, G
使用荧光滴定、超速离心测量和快速动力学技术研究了来自大肠杆菌的单链 DNA 结合蛋白（ssB 蛋白）与寡核苷酸和单链 DNA 之间的复合物形成。 oligo(dT)-ssB 复合物的化学计量测定表明，ssB 蛋白的 4 个亚基中的每一个都代表约 8 个残基长的寡核苷酸的结合位点。寡聚 (dT) 或多聚 (dT) 占据所有 4 个结合位点会导致内在蛋白质荧光 80% 猝灭。结合位点几乎是等效且独立的。对于d(pT)16，内在结合常数是6倍。 105 M-1，并且对于足够长以在ssB四聚体上连续延伸的d(pT)30-40，结合常数高于5倍。 108 M-1。寡腺苷酸的结合比相应的寡 (dT) 种类弱约 2 个数量级。与oligo(dA)-ssB复合物的形成相比，oligo(dT)的结合对离子强度的依赖性非常弱。对于 d(pT)8、d(pT)16 和 d(pT)30-40，复合物的形成可以通过简单的 1 步反应来描述。相互作用的强度主要用解离速率常数来表示。在与聚（dT）或聚（dA）的协同复合物中，ssB四聚体上的所有4个结合位点也被占据。单链 DNA 盘绕在 ssB 分子周围。复合物的荧光熔解实验表明，单链DNA的构象对复合物的稳定性有很大影响。
The complex formation between the single-strand DNA binding protein (ssB protein) from E. coli and oligonucleotides and single-stranded DNA was studied using fluorescence titrations, ultracentrifugation measurements and fast kinetic techniques. Determination of the stoichiometries of oligo(dT)-ssB complexes shows that each of the 4 subunits of the ssB protein represents a binding site for an oligonucleotide about 8 residues long. Occupation of all 4 binding sites with oligo(dT) or poly(dT) leads to 80% quenching of the intrinsic protein fluorescence. The binding sites are nearly equivalent and independent. For d(pT)16, the intrinsic binding constant is 6 .times. 105 M-1, and for d(pT)30-40, which is long enough to extend continuously over the ssB tetramer, the binding constant is higher than 5 .times. 108 M-1. Oligoadenylates bind about 2 orders of magnitude weaker than the corresponding oligo(dT) species. The binding of oligo(dT) is very weakly dependent on ionic strength, in contrast to the oligo(dA)-ssB complex formation. For d(pT)8, d(pT)16, and d(pT)30-40, the complex formation can be described by a simple 1-step reaction. The strength of the interaction is mainly expressed in the rate constant is dissociation. In the cooperative complexes with poly(dT) or poly(dA), all 4 binding sites on the ssB tetramer are also occupied. Single-stranded DNA is coiled around the ssB molecule. Fluorescence melting experiments of the complexes show that the conformation of the single-stranded DNA has a strong influence on the stability of the complexes.