Assembly of Xenopus transcription factor III A-5S RNA complex.
Assembly of Xenopus transcription factor III A-5S RNA complex.
复制标题
非洲爪蟾转录因子 III A-5S RNA 复合物的组装。
作者：
Callaci,TP;Cai,GZ;Lee,JC;Daly,TJ;Wu,CW
托马斯P. Callaci，** Guang-Zuan Cai，* James C. Lee，1991，托马斯J. Daly，§和Cheng-wen Wu 8，11 E. A. Doisy Department of Biochemistry，St. Louis University School of Medicine，St. Louis，密苏里州63104，and Department of Pharmacological Science，州立大学of纽约斯托尼布鲁克，斯托尼布鲁克，纽约11794接收日期：1989年11月21日;修订版手册接收日期：1990年1月19日摘要：非洲爪蟾5S rRNA基因表达的调控涉及多种蛋白质因子，其中转录因子Ⅲ A（TFIIIA）是最重要的转录因子之一。该因子可以作为蛋白质-RNA复合物分离。在pH7.5和23 ℃条件下，用沉降速率法和凝胶电泳法研究了该复合物的组装行为.通过260 nm处的吸光度监测反应边界;因此，边界的形状主要反映RNA或含RNA的复合物。重均沉降系数（f20 w）的值随蛋白质-RNA浓度而变化。在低浓度下，J20> w的值随着浓度的增加而增加。无限稀释时S20 w的外推值为5.2 S，与相同实验条件下游离非洲爪蟾rRNA的外推值相同。此外，在特定RNA浓度下，通过稀释浓缩样品或浓缩稀释的样品可以获得相同的S20 w值。这些结果表明，复合物的形成可以用可逆过程来描述。当通过计算机拟合分析数据时，拟合沉降数据的最简单模型是TFIIIA和RNA形成1：1复合物，其自聚集成二聚体。PR和（PR）2的沉降系数（j20 w）分别为7.5和10.6 S，其中PR和（PR）2分别为1：1的TFIIIA-RNA复合物及其二聚体。蛋白质-RNA相互作用也通过凝胶电泳进行了研究。通过蛋白质和RNA在单一凝胶上的差异染色来鉴定分离的组分。除了蛋白质和核酸染色的两条带外，还检测到一条对应于游离5S rRNA的带。这两个蛋白质-RNA复合物的质量比被确定为2.3，这意味着一个复合物可能是另一个的二聚体。这些电泳结果表明，在低浓度下，TFIIIA和RNA以单独的游离实体存在。在较高浓度下，它们形成二聚化的复合物。这些结果与沉积作用的结果完全一致。总之，非洲爪蟾TFIIIA-RNA复合物经历大分子组装的可逆平衡，其特征在于TFIIIA+ RNA<= s TFIIIA-RNA复合物（TF-
Thomas P. Callaci,** Guang-Zuan Cai,* James C. Lee,*’1 Thomas J. Daly, § and Cheng-wen Wu8, 11 E. A. Doisy Department of Biochemistry, St. Louis University School of Medicine, St. Louis, Missouri 63104, and Department of Pharmacological Science, State University of New York at Stony Brook, Stony Brook, New York 11794 Received November 21, 1989; Revised Manuscript Received January 19, 1990 abstract: The regulation of Xenopus 5S rRNA gene expression involves multiple protein factors, among which is transcription factor III A (TFIIIA). This factor can be isolated as a protein-RNA complex. The assembly behavior of this complex was studied by sedimentation velocity and gel electrophoresis at pH 7.5 and 23 C. The reaction boundary was monitored by the absorbance at260 nm; thus, the shape of the boundary reflects mainly RNA or RNA-containing complexes. Values for the weight-average sedimentation coefficient (f20 w) change with protein-RNA concentration. At low concentrations, values of J20> w increase with increasing concentration. The extrapolated value for S20 w at infinite dilution is 5.2 S, the same value for free Xenopus rRNA under the same experimentation conditions. Furthermore, the same value of S20 w at a specific RNA concentration can be obtained either by dilution of a concentrated sample or by con-centrating a diluted one. These results indicate that complex formation can be described by a reversible process. When the data are analyzed by computer fitting, the simplest model that fits the sedimentation data is that TFIIIA and RNA form a 1: 1 complex which self-aggregates to a dimer. The sedimentation coefficients (j20w) of PR and (PR) 2 are 7.5 and 10.6 S, respectively, where PR and (PR) 2 are the 1: 1 TFIIIA-RNA complex and its dimer, respectively. The protein-RNA interaction was also investigated by gel electrophoresis. The resolved components were identified by differential staining for protein andRNA on a single gel. One band corresponding to free 5S rRNA was detected in addition to two bands which stained for both protein and nucleic acids. The mass ratio of these two protein-RNA complexes was determined to be 2.3, implying that one complex is probably a dimer of the other. These electrophoretic results indicatethat at low concentration TFIIIA and RNA exist as individual free entities. At higher concentration, they form a complex which dimerizes. These results are totally consistent with that of sedimentation. In summary, the Xenopus TFIIIA-RNA complex undergoes a reversible equilibrium of macromolecular assembly which is characterized as TFIIIA+ RNA<= s TFIIIA-RNA complex(TF-