Cross-links between ribosomal proteins of 30S subunits in 70S tight couples and in 30S subunits.
Cross-links between ribosomal proteins of 30S subunits in 70S tight couples and in 30S subunits.
复制标题
70S 紧密偶和 30S 亚基中 30S 亚基的核糖体蛋白之间的交联。
DOI：
10.1021/bi00285a029
复制
发表时间：
1983
期刊：
影响因子：
2.9
通讯作者：
Traut,RR
中科院分区：
文献类型：
--
作者：
Lambert,JM;Boileau,G;Cover,JA;Traut,RR
John M. Lambert，* Guy Boileau，5 Jan A.封面，罗伯特R。Traut* 摘要：核糖体70 S紧偶和30 S亚基从他们衍生的2-亚氨基硫杂环戊烷修饰的条件下，每个蛋白质约两个巯基基团被添加到核糖体颗粒。70 S和30 S颗粒没有用升高浓度的NH 4Cl处理，与早期研究中使用的那些相反。修饰后的颗粒被氧化以促进二硫键的形成。通过使用排除二硫键交换的条件从交联颗粒中提取蛋白质。二硫键连接的蛋白质复合物进行分馏的基础上，在聚丙烯酰胺/尿素凝胶在pH 5.5的电泳电荷。通过二维对角聚丙烯酰胺/十二烷基硫酸钠凝胶电泳分析来自尿素凝胶连续切片的蛋白质。交联复合物中蛋白质的最终鉴定是通过蛋白质的放射性碘标记，然后通过二维聚丙烯酰胺/尿素凝胶电泳进行的。人们的注意力集中在30 S蛋白之间的交联。我们报告的27个交联的二聚体和2个三聚体的30 S蛋白的鉴定，但其中一个都被发现在70 S核糖体和游离30 S亚基在类似的产量。当使用2-亚氨基硫杂环戊烷时，先前未报道其中7个交联，S3-S13、S13-S21、S14-S19、S7-S12、S9-S13、S11-S21和S6-S18-S21。首次报道了交联S3-S13、S13-S21、S7-S12、S11-S21和S6-S18-S21。七个新的交叉链接的识别说明和详细讨论。在Sommer &Traut（1976）的早期研究中报道的十种二聚体[Sommer，A.，& Traut，RR（1976）J. Mol. Biol.106，995-1015]，使用用高盐浓度处理的30 S亚基，在本文报道的实验中没有发现。30 S核糖体亚基的三维结构已经通过多种技术进行了广泛的研究（Brimacombe等人，1978年）。蛋白质交联（Traut等人，1980; Expert-Bezan 9 on等人，1977; Sommer & Traut，1976）、免疫电子显微术（Lake，1980; Stoffler等人，1980; Kahan等人，1981）和中子散射技术（Langer等人，1978; Ramakrishnan等人，1981）已被用于定义30 S亚基中的核糖体蛋白质排列。利用这些证据已经建立了初步的模型。不同的实验方法所得的结果有很好的一致性.少量的交联似乎与免疫电子显微镜不一致（Kahan等人，1981）和中子散射模型（Ramakrishnan等人，1981年）。游离30 S亚基在与50 S亚基缔合后经历镁浓度依赖性构象变化（Ball等人，1973），其需要能量来克服活化势垒（Zamir等人，1971年）。化学修饰已被用作亚基缔合后构象变化的探针。几项研究已经表明，一些蛋白质通过亚基缔合而被保护免于修饰，而其他蛋白质与游离亚基相比在70 S核糖体中变得更具反应性（Huang & Cantor，1972; Litman et al. 1976年）。通过使用鸟嘌呤特异性试剂kethoxal，在30 S亚基的16 S RNA中的特异性位点获得了类似的结果（Herr等人，1979年）。中子散射结果表明，30 S亚基在70 S核糖体中具有与游离30 S亚基相同的总体形状
John M. Lambert,* Guy Boileau, 5 Jan A. Cover, and Robert R. Traut* abstract: Ribosome 70S tight couples and 30S subunits derived from them were modified with 2-iminothiolane under conditions where about two sulfhydryl groups per protein were added to the ribosomal particles. The 70S and 30S particles were not treated with elevated concentrations of NH4C1, in contrast to those used in earlier studies. The modified particles were oxidized to promote disulfide bond formation. Proteins were extracted from the cross-linked particles by using con-ditions to precludedisulfide interchange. Disulfide-linked protein complexes were fractionated on the basis of charge by electrophoresis in polyacrylamide/urea gels at pH 5.5. The proteins from sequential slices of the urea gels were analyzed by two-dimensional diagonal polyacrylamide/sodium dodecyl sulfate gel electrophoresis. Final identification of proteins in cross-linked complexes was made byradioiodination of the proteins, followed by two-dimensional polyacrylamide/urea gel electrophoresis. Attention was focused on cross-links be-tween 30S proteins. We report the identification of 27 cross-linked dimers and 2 trimers of 30S proteins, all but one of which were found in both 70S ribosomes and free 30S subunits in similar yield. Seven of the cross-links, S3-S13, S13-S21, S14-S19, S7-S12, S9-S13, S11-S21, and S6-S18-S21, have not been reported previously when 2-iminothiolane was used. Cross-links S3-S13, S13-S21, S7-S12, SI 1-S21, and S6-S18-S21 are reported for the first time. The identification of the seven new cross-links is illustrated and discussed in detail. Ten of the dimers reported in the earlier studies of Sommer &Traut (1976)[Sommer, A., & Traut, RR (1976) J. Mol. Biol. 106, 995-1015], using 30S subunits treated with high salt concentrations, were not found in the experiments reported here. e three-dimensional structure of the 30S ribosomal subunit has been investigated extensively by a variety of techniques (Brimacombe et al., 1978). Protein cross-linking (Traut et al., 1980; Expert-Bezan9on et al., 1977; Sommer & Traut, 1976), immune electron microscopy (Lake, 1980; Stoffler et al., 1980; Kahan et al., 1981), and neutron scattering techniques (Langer et al., 1978; Ramakrishnan et al., 1981) have been used to define ribosomal protein arrangement in the 30S subunit. Preliminary models have been constructed by using this evidence. There is goodgeneral agreement of the results from the different experimental approaches. A smallnumber of cross-links have appeared inconsistent with immune electron microscopy (Kahan et al., 1981) and with the neutron scat-tering model (Ramakrishnan et al., 1981). Free 30S subunits undergo a magnesium concentration dependent conformational change upon association with 50S subunits (Ball et al., 1973), which requires energy to overcome an activation barrier (Zamir et al., 1971). Chemical modi-fication has been used as a probe of conformational change upon subunit association. Several studies have shown that some proteins are protected from modification by subunit association, while others become more reactive in the 70S ribosome compared to free subunits (Huang & Cantor, 1972; Litman et al., 1976). Similar results have been obtained at specific sites in the 16S RNA of the 30S subunit by using the guanine-specific reagent kethoxal (Herr et al., 1979). Neutron scattering results indicate that the30S subunit has the same general shape in 70S ribosomes and as free 30S subunits