Renaturation of complementary DNA strands mediated by purified mammalian heterogeneous nuclear ribonucleoprotein A1 protein: implications for a mechanism for rapid molecular assembly.

Purified heterogeneous nuclear ribonucleoprotein (hnRNP) A1 protein, which is found in vivo associated with heterogeneous nuclear RNA (hnRNA), promotes the rapid renaturation of nucleic acid strands. Maximal renaturation activity requires the glycine-rich carboxyl-terminal one-third of the protein, although the amino-terminal two-thirds also has activity. The A1-mediated reaction is second-order with respect to complementary DNA concentration, and the renaturation rate constant at 37 degrees C with A1 is about 3000-fold greater than in the absence of the protein. At 60 degrees C, the A1-mediated renaturation rate is even faster, and is about 300-fold greater than protein-free reactions carried out at 68 degrees C in 1 M NaCl. Provided that sufficient A1 protein is present to coat all strands in solution, the presence of nonhomologous, single-stranded DNA does not significantly inhibit the reaction. Moreover, renaturation of short strands to their complement contained in very long strands is nearly as efficient as between two short strands. These results indicate that A1 may be useful for procedures that rely on nucleic acid renaturation. We propose that A1 promotes rapid renaturation primarily by reducing the entropic barrier of bimolecular strand association through relatively transient interactions between A1-coated strands. Such interactions, mediated by flexible repeating domains, may act generally to increase the association kinetics of highly specific molecular assemblies in processes such as RNA maturation, transcription, translation, and transport. IMAGES:

纯化的异质核核糖核蛋白（heterogeneous nuclear ribonucleoprotein, hnRNP）A1蛋白在体内可与异质核RNA（heterogeneous nuclear RNA, hnRNA）结合，能够促进核酸链的快速复性。该蛋白复性活性的最大化依赖于其富含甘氨酸的羧基末端三分之一区段，即便氨基末端三分之二区段同样具备复性活性。A1介导的复性反应速率与互补DNA浓度呈二级动力学关系，在37℃条件下，存在A1蛋白时的复性速率常数较无蛋白对照组高出约3000倍。在60℃时，A1介导的复性速率进一步提升，较1M NaCl体系中68℃下的无蛋白复性反应快约300倍。当体系中存在足量A1蛋白以包被溶液中所有核酸链时，非同源单链DNA的存在不会对该反应产生显著抑制作用。此外，短链核酸与长链核酸内其互补序列的复性效率，几乎与两条短链核酸之间的复性效率相当。上述结果表明，A1蛋白可应用于依赖核酸复性的实验操作流程。我们推测，A1主要通过A1包被的核酸链之间的相对短暂相互作用，降低双分子链结合过程中的熵垒，从而实现快速复性。这种由柔性重复结构域介导的相互作用，或可普遍提升高度特异性分子组装过程的结合动力学效率，相关过程涵盖RNA成熟、转录、翻译以及物质转运等。IMAGES: