McAfee J G, Soltaninassab S R, Lindsay M E, LeStourgeon W M
Department of Molecular Biology, Vanderbilt University, Nashville, Tennessee 37235, USA.
Biochemistry. 1996 Jan 30;35(4):1212-22. doi: 10.1021/bi951974k.
Proteins C1 and C2 together comprise about one-third the protein mass of mammalian core 40S heterogeneous nuclear ribonucleoprotein particles (40S hnRNP) and exist as heterotetramers of (C1)3C2. On the basis of nonequilibrium binding studies, it has been suggested that the C proteins specifically bind oligo(U)- and poly(U)-rich sequences, and preferentially associate with uridine-rich regions near the 3' termini of many introns. We describe here a more quantitative characterization of the equilibrium binding properties of native and recombinant C protein to homoribopolymers using fluorescence spectroscopy. Like C protein from HeLa cells, the recombinant proteins spontaneously oligomerize to form tetramers with the same hydrodynamic properties as native protein. Near-stoichiometric binding titrations of the fluorescent homoribopolymer polyethenoadenosine (poly[r(epsilon A)]) with recombinant (C1)4 and (C2)4 homotetramers along with competition binding assays with poly(A) and poly(C) indicate that the binding site size (n) is between 150 and 230 nucleotides. This site size range is in close agreement with that previously determined for native C protein through hydrodynamic and ultrastructural studies (approximately 230 nucleotides). (C1)4 and (C2)4 bind poly(G) with intrinsic affinities (Ki) of 10(9) M-1, which are a hundredfold higher than their affinities for poly(U). In opposition to reports that C protein does not bind poly(A) and poly(C), we find that the C proteins bind these substrates with moderate Ki, but with high cooperativity (omega). The overall affinity (K omega) for the binding of both proteins to poly(A) and poly(C) is 10-fold higher (> 10(8) but < 10(9) M-1) than their affinities for poly(U). The highly cooperative binding of C protein to these substrates provides a mechanistic basis for the distribution of C protein along the length of nucleic acid substrates.
蛋白质C1和C2共同构成了哺乳动物核心40S异质核糖核蛋白颗粒（40S hnRNP）约三分之一的蛋白质质量，以（C1）3C2的异源四聚体形式存在。基于非平衡结合研究，有人提出C蛋白特异性结合富含寡聚（U）和多聚（U）的序列，并优先与许多内含子3'末端附近富含尿苷的区域结合。我们在此描述了使用荧光光谱法对天然和重组C蛋白与同聚核糖核酸聚合物的平衡结合特性进行的更定量表征。与来自HeLa细胞的C蛋白一样，重组蛋白自发寡聚形成四聚体，其流体动力学性质与天然蛋白相同。用重组（C1）4和（C2）4同型四聚体对荧光同聚核糖核酸聚合物聚乙二醛腺苷（poly[r(εA)]）进行近化学计量结合滴定，以及用聚（A）和聚（C）进行竞争结合试验表明，结合位点大小（n）在150至230个核苷酸之间。这个位点大小范围与先前通过流体动力学和超微结构研究确定的天然C蛋白的位点大小范围（约230个核苷酸）非常一致。（C1）4和（C2）4以10^9 M^-1的内在亲和力（Ki）结合聚（G），这比它们对聚（U）的亲和力高一百倍。与关于C蛋白不结合聚（A）和聚（C）的报道相反，我们发现C蛋白以中等的Ki但高协同性（ω）结合这些底物。两种蛋白与聚（A）和聚（C）结合的总体亲和力（Kω）比它们对聚（U）的亲和力高10倍（>10^8但<10^9 M^-1）。C蛋白与这些底物的高度协同结合为C蛋白沿核酸底物长度的分布提供了一个机制基础。
