Setzer D R, Brown D D
J Biol Chem. 1985 Feb 25;260(4):2483-92.
5 S ribosomal RNA in Xenopus has been shown to be transcribed in vitro from 5 S RNA genes that remain stably associated with required transcription factors through multiple rounds of transcription (Bogenhagen, D. F., Wormington, W. M., and Brown, D. D. (1982) Cell 28, 413-421). We have studied the formation and stability of these "transcription complexes" by using cloned 5 S RNA genes immobilized on cellulose as templates for the assembly of complexes in crude extracts. RNA polymerase III is the least tightly bound component required for transcription of 5 S RNA genes. All other factors remain bound in 1 M NaCl, even though transcription complexes do not form at salt concentrations as low as 0.25 M. RNA polymerase III dissociates from transcription complexes as a result of RNA synthesis and is capable of reassociating with complexes to support additional rounds of transcription. A 5 S-specific positive transcription factor (factor A) and two crude phosphocellulose column fractions (B and C) are also required for 5 S RNA synthesis in vitro (Engelke, D. R., Ng, S.-Y., Shastry, B. S., and Roeder, R. G. (1980) Cell 19, 717-728; Segall, J., Matsui, T., and Roeder, R. G. (1980) J. Biol. Chem. 255, 11986-11991; Shastry, B. S., Ng, S.-Y., and Roeder, R. G. (1982) J. Biol. Chem. 257, 12979-12986). Fraction B stably interacts with 5 S RNA genes to form a stable, active complex only after the template has first been incubated with factor A and fraction C. In contrast, either factor A or fraction C can stably associate with 5 S RNA genes in the absence of other factors. The activities of fractions B and C are removed from solution as a result of transcription complex formation, suggesting the factors in these fractions act stoichiometrically. The rate-limiting step in complex formation is carried out by fraction B, which accounts for the lag in transcription activity observed in crude extracts.
非洲爪蟾的5S核糖体RNA已被证明可在体外从5S RNA基因转录而来，这些基因在多轮转录过程中与所需转录因子保持稳定结合（博根哈根，D.F.，沃明顿，W.M.，以及布朗，D.D.（1982年）《细胞》28卷，413 - 421页）。我们通过使用固定在纤维素上的克隆5S RNA基因作为模板，在粗提物中组装复合物，研究了这些“转录复合物”的形成和稳定性。RNA聚合酶III是5S RNA基因转录所需的结合最不紧密的成分。所有其他因子在1M NaCl中仍保持结合状态，尽管在低至0.25M的盐浓度下转录复合物无法形成。由于RNA合成，RNA聚合酶III从转录复合物中解离，并且能够与复合物重新结合以支持额外的转录轮次。体外合成5S RNA还需要一种5S特异性正转录因子（因子A）以及两个粗制磷酸纤维素柱层析组分（B和C）（恩格尔克，D.R.，吴，S.-Y.，沙斯特里，B.S.，以及罗德，R.G.（1980年）《细胞》19卷，717 - 728页；西格尔，J.，松井，T.，以及罗德，R.G.（1980年）《生物化学杂志》255卷，11986 - 11991页；沙斯特里，B.S.，吴，S.-Y.，以及罗德，R.G.（1982年）《生物化学杂志》257卷，12979 - 12986页）。只有在模板首先与因子A和组分C孵育后，组分B才会与5S RNA基因稳定相互作用形成稳定的活性复合物。相比之下，在没有其他因子的情况下，因子A或组分C都可以与5S RNA基因稳定结合。由于转录复合物的形成，组分B和C的活性从溶液中去除，这表明这些组分中的因子按化学计量起作用。复合物形成中的限速步骤由组分B执行，这解释了在粗提物中观察到的转录活性延迟现象。
