STRUCTURE AND MACROMOLECULAR INTERACTIONS OF 5S RNA .1. STOICHIOMETRY, COOPERATIVITY, AND STABILITY OF INTERACTIONS BETWEEN 5S RNA AND PROTEINS L5, L18, AND L25 FROM 50S RIBOSOMAL-SUBUNIT OF ESCHERICHIA-COLI
STRUCTURE AND MACROMOLECULAR INTERACTIONS OF 5S RNA .1. STOICHIOMETRY, COOPERATIVITY, AND STABILITY OF INTERACTIONS BETWEEN 5S RNA AND PROTEINS L5, L18, AND L25 FROM 50S RIBOSOMAL-SUBUNIT OF ESCHERICHIA-COLI
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DOI：
10.1021/bi00606a002
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发表时间：
1978-01-01
期刊：
影响因子：
2.9
通讯作者：
ZIMMERMANN, RA
中科院分区：
文献类型：
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作者：
SPIERER, P;ZIMMERMANN, RA
E.通过多种标准评估具有50 S核糖体亚基蛋白L5、L18和L25的大肠杆菌。复合物形成对TMK缓冲液（50 mM Tris-HCl（pH 7.6）-20 mM MgCl 2 -300 mM KCl）中蛋白质和RNA浓度的依赖性表明，3种蛋白质对核酸的亲和力显著不同。在过量蛋白质存在下的缔合的化学计量的测量揭示，饱和时L5、L18和L25的蛋白质：RNA结合摩尔比分别为0.6：1、1.1：1和0.7：1。因此，RNA分子中每种蛋白质的特异性连接位点不超过1个。改变溶液条件以评估pH、Mg 2+浓度和K+浓度对相互作用的稳定性的影响。L5-5S RNA复合物在pH 6.5-9，[Mg ~（2+）]为10-20 mM，[K ~+]为300 - 400 mM时观察到最佳结合; L18-5S RNA复合物在pH 7.5-9，[Mg ~（2+）]为10-20 mM，[K ~+]为100-200 mM时观察到最佳结合;对于L25-5S RNA复合物，pH 7.5-9，[Mg 2 +]为0.3-20 mM，[K+]为200-300 mM。在一系列单独的实验中，L5和TMK缓冲液的结合被L18协同刺激，L18的组分浓度比L5结合所需的浓度低大约10倍一个人这两种蛋白质相互影响的另一个也清楚地表现在涉及pH值和离子环境的变化的测定。协同模式表明，在5S RNA中的L5和L18的结合位点在功能上彼此相关，但与蛋白L25的结合位点不同。
Interactions of 5S RNA from E. coli with 50S ribosomal subunit proteins L5, L18 and L25 were evaluated by a number of criteria. The dependence of complex formation on protein and RNA concentration in TMK buffer (50 mM Tris-HCl (pH 7.6)-20 mM MgCl2-300 mM KCl) indicated that the 3 proteins differ substantially in their affinity for the nucleic acid. Measurement of the stoichiometry of association in the presence of excess protein revealed that molar protein:RNA binding ratios for L5, L18 and L25 at saturation were 0.6:1, 1.1:1 and 0.7:1, respectively. The RNA molecule therefore contains no more than 1 specific site of attachment for each of the proteins. Solution conditions were varied to assess the effects of pH, Mg2+ concentration and K+ concentration on the stability of the interactions. Optimal binding was observed for the L5-5S RNA complex at pH 6.5-9, [Mg2+] of 10-20 mM and [K+] of 300 to 400 mM; for the L18-5S RNA complex at pH 7.5-9, [Mg2+] of 10-20 mM and [K+] of 100-200 mM; and for the L25-5S RNA complex at pH 7.5-9, [Mg2+] of 0.3-20 mM and [K+] of 200-300 mM. In a separate series of experiments, the association of L5 and TMK buffer was cooperatively stimulated by L18 at component concentrations roughly tenfold less than were required for the association of L5 alone. The mutual influence of these 2 proteins upon one another was also clearly manifested in assays involving variation of pH and ionic environment. The pattern of cooperativity showed that the binding sites for L5 and L18 in the 5S RNA are functionally related to each other, but distinct from that for protein L25.