INTERACTIONS OF BACTERIOPHAGE T4-CODED GENE 32 PROTEIN WITH NUCLEIC-ACIDS .1. CHARACTERIZATION OF THE BINDING INTERACTIONS
INTERACTIONS OF BACTERIOPHAGE T4-CODED GENE 32 PROTEIN WITH NUCLEIC-ACIDS .1. CHARACTERIZATION OF THE BINDING INTERACTIONS
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DOI：
10.1016/0022-2836(81)90335-1
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发表时间：
1981-01-01
影响因子：
5.6
通讯作者：
VONHIPPEL, PH
中科院分区：
文献类型：
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作者：
KOWALCZYKOWSKI, SC;LONBERG, N;VONHIPPEL, PH
研究了噬菌体t4编码基因32蛋白与寡核苷酸和多核苷酸相互作用的分子细节。基因32蛋白2-8个核苷酸残基中长度为(l)的寡核苷酸的结合亲和力（Koligo）基本上与碱基组成或糖类型无关。这种结合不受盐浓度和寡核苷酸长度的影响；在Koligo中甚至没有观察到预期的统计长度因子，这表明结合发生在寡核苷酸晶格的末端，并且寡核苷酸不能自由地穿过结合位点。基因32蛋白与多核苷酸的合作（连续）或分离结合是非常不同的：这种结合是高度依赖盐的（.delta）。日志K.omega. / .delta。log [NaCl] .simeq。-7)，基本上是盐浓度下的化学计量学。0.2 M（聚（rA））。在较高的盐浓度下，蛋白质的结合变得更弱，结合等温线表现出典型的合作（s形）。通过将不同盐浓度下的协同结合等温线拟合到理论图中，并在极低的蛋白质结合密度（.nu.）下测量结合，证明了K.omega的完全盐依赖性。为本征结合常数(K)；协同性参数（.omega.）基本上与盐浓度无关。通过测定含有一系列不同阴离子和阳离子的盐存在下的滴定曲线，表明基因32蛋白-多核苷酸相互作用的盐依赖性主要是由于阴离子（而不是阳离子）位移效应。长度足以结合2个或更多基因32蛋白单体的寡核苷酸的结合参数表现出介于寡核苷酸和多核苷酸结合模式之间的行为。这些不同的结合模式可能反映了蛋白质的不同构象；对这些结果进行分析，得出基因32蛋白在不同结合模式下与核酸相互作用的初步分子模型。
Molecular details of the interaction of bacteriophage T4-coded gene 32 protein with oligo- and polynucleotides are examined. The binding affinity (Koligo) of oligonucleotides of length (l) from 2-8 nucleotide residues for gene 32 protein is essentially independent of base composition or sugar type. This binding shows little dependence on salt concentration and on oligonucleotide length; even the expected statistical length factor in Koligo is not observed, suggesting that binding occurs at the end of the oligonucleotide lattice and that the oligonucleotide is not free to move across the binding site. Cooperative (contiguous) or isolated binding of gene 32 protein to polynucleotides is very different: here binding is highly salt dependent (.delta. log K.omega./.delta. log [NaCl] .simeq. -7) and essentially stoichiometric at salt concentrations .apprx. 0.2 M (for poly(rA)). Binding becomes much weaker and the binding isotherms appear typically cooperative (sigmoid) in protein concentration at higher salt concentrations. By fitting the cooperative binding isotherms to theoretical plots at various salt concentrations and by measuring binding at very low protein binding density (.nu.), it is demonstrated that the entire salt dependence of K.omega. is in the intrinsic binding constant (K); the cooperativity parameter (.omega.) is essentially independent of salt concentration. By determining titration curves in the presence of salts containing a series of different anions and cations, it shown that the major part of the salt dependence of the gene 32 protein-polynucleotide interaction is due to anion (rather than to cation) displacement effects. Binding parameters of oligonucleotides of length sufficient to bind 2 or more gene 32 protein monomers show behavior intermediate between the oligonucleotide and the polynucleotide binding modes. These different binding modes probably reflect different conformations of the protein; the results are analyzed to produce a preliminary molecular model of the interactions of gene 32 protein with nucleic acids in its different binding modes.