Characterization of the DNA-binding domain of beta protein, a component of phage lambda Red-pathway by UV catalyzed cross-linking
Characterization of the DNA-binding domain of beta protein, a component of phage lambda Red-pathway by UV catalyzed cross-linking
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DOI：
10.1016/s0378-1119(96)00518-5
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发表时间：
1996-12-05
期刊：
影响因子：
3.5
通讯作者：
Muniyappa, K
中科院分区：
文献类型：
--
作者：
Mythili, E;Kumar, KA;Muniyappa, K
β蛋白是噬菌体lambda红色通路的关键成分，在重组缺陷突变株中对其生长和一般的基因重组是必需的。为了便于结构-功能关系的研究，我们过表达了β蛋白，并将其纯化为均一。化学交联剂戊二醛用于稳定溶液中β蛋白的物理缔合。经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳法分离得到一条67 kDa的条带，与均二聚体对应。化学计量测量表明，β蛋白的1个单体/5个核苷酸残基的位置大小。凝胶迁移率变化分析表明，β蛋白与36聚体形成稳定的核蛋白复合体，但不与27或17聚体DNA形成稳定的核蛋白复合体。有趣的是，β蛋白与DNA的相互作用和核蛋白复合体的稳定性依赖于氯化镁的存在，250 mM的氯化钠取消了这种结合。β蛋白与36-聚DNA结合的K-d约为1.8×10(-6)M。用凝乳酶产生的天然β蛋白或其片段与36-聚DNA进行光化学交联以确定其DNA结合区。对交联肽的表征表明，DNA结合所需的氨基酸位于N末端的20 kDa多肽中。这些发现为进一步了解β蛋白的结构和功能提供了基础。
beta protein, a key component of Red-pathway of phage lambda is necessary for its growth and general genetic recombination in recombination-deficient mutants of Escherichia coli. To facilitate studies on structure-function relationships, we overexpressed beta protein and purified it to homogeneity. A chemical cross-linking reagent, glutaraldehyde, was used to stabilize the physical association of beta protein in solution. A 67-kDa band, corresponding to homodimer, was identified after separation by SDS-polyacrylamide gel electrophoresis. Stoichiometric measurements indicated a site-size of 1 monomer of beta protein/5 nucleotide residues. Electrophoretic gel mobility shift assays suggested that beta protein formed stable nucleoprotein complexes with 36-mer, but not with 27- or 17-mer DNA. Interestingly, the interaction of beta protein with DNA and the stability of nucleoprotein complexes was dependent on the presence of MgCl2, and the binding was abolished by 250 mM NaCl. The K-d of beta protein binding to 36-mer DNA was on the order of 1.8 x 10(-6) M. Photochemical cross-linking of native beta protein or its fragments, generated by chymotrypsin, to 36-mer DNA was performed to identify its DNA-binding domain. Characterization of the cross-linked peptide disclosed that amino acids required for DNA-binding specificity resided within a 20-kDa peptide at the N-terminal end. These findings provide a basis for further understanding oi the structure and function of beta protein.