INTERACTION OF NUCLEOLAR PHOSPHOPROTEIN-B23 WITH NUCLEIC-ACIDS
INTERACTION OF NUCLEOLAR PHOSPHOPROTEIN-B23 WITH NUCLEIC-ACIDS
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DOI：
10.1021/bi00450a037
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发表时间：
1989-11-28
期刊：
影响因子：
2.9
通讯作者：
OLSON, MOJ
中科院分区：
文献类型：
--
作者：
DUMBAR, TS;GENTRY, GA;OLSON, MOJ
通过凝胶阻滞和滤光片结合测定、荧光技术和圆二色性检查真核核仁磷蛋白 B23 与核酸的相互作用。所有研究均利用通过新开发的纯化程序在天然条件下制备的蛋白质。使用噬菌体 M13 DNA 进行的电泳凝胶迁移率变动分析表明蛋白 B23 是单链核酸结合蛋白。这在天然或热变性线性化质粒 pUC18 DNA 的竞争结合测定中得到证实，其中蛋白质显示出对变性形式的明显偏好。在其他竞争测定中，单链合成核糖核苷酸与脱氧核糖核苷酸没有明显的偏好。与聚(核糖乙烯腺苷酸)的平衡结合表明具有11个核苷酸的蛋白质结合位点大小和5×的表观结合常数(KΩ)的协同配体结合。 107 M-1，其包括6.3倍的内在结合常数(K)。 104 M-1，协同因子 (Ω) 为 800。在圆二色性 (CD) 研究中，蛋白质 B23 当与单链合成核酸 Poly(rA) 和 Poly(rC) 结合时，会导致椭圆率降低，并使 260-270 nm 处的正峰向更高波长移动，表明螺旋不稳定活性。双链聚(dA.cntdot.dT)没有观察到CD变化。添加蛋白质后，poly(rA) 椭圆率的变化呈 S 形，证实了荧光方法所观察到的协同行为。这些研究表明，蛋白质 B23 以高亲和力与单链核酸协同结合，并表现出 RNA 螺旋不稳定活性。这些特征可能与其在核糖体组装中的作用有关。
The interaction of eukaryotic nucleolar phosphoprotein B23 with nucleic acids was examined by gel retardation and filter binding assays, by fluoroscence techniques, and by circular dichroism. All studies utilized protein prepared under native conditions by a newly developed purification procedure. Electrophoretic gel mobility shift assays with phage M13 DNA suggested that protein B23 is a single-stranded nucleic acid binding protein. This was confirmed in competition binding assays with native or heat-denatured linearized plasmid pUC18 DNA where the protein showed a marked preference for the denatured form. In other competition assays, there was no apparent preference for single-stranded synthetic ribo- versus deoxyribonucleotides. Equilibrium binding with poly(riboethenoadenylic acid) indicated cooperative ligand binding with a protein binding site size of 11 nucleotides and an apparent binding constant (K.omega.) of 5 .times. 107 M-1 which includes an intrinsic binding constant (K) of 6.3 .times. 104 M-1 and a cooperativity factor (.omega.) of 800. In circular dichroism (CD) studies, protein B23, when combined with the single-stranded synthetic nucleic acids poly(rA) and poly(rC), effected a decrease in ellipticity and a shift of the positive peak at 260-270 nm toward higher wavelengths, indicating helix destabilizing activity. No CD changes were seen with double-stranded poly(dA .cntdot. dT). The change in ellipticity of poly(rA) was sigmoidal upon addition of protein, confirming the cooperative behavior seen with fluorescence methods. These studied indicate that protein B23 binds cooperatively with high affinity for single-stranded nucleic acids and exhibits RNA helix destabilizing activity. These features may be related to its role in ribosome assembly.