Kinetics of the RNA-DNA helicase activity of Escherichia coli transcription termination factor rho. 1. Characterization and analysis of the reaction.
Kinetics of the RNA-DNA helicase activity of Escherichia coli transcription termination factor rho. 1. Characterization and analysis of the reaction.
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大肠杆菌转录终止因子 rho 的 RNA-DNA 解旋酶活性的动力学。
DOI：
10.1021/bi963179s
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发表时间：
1997
期刊：
影响因子：
--
通讯作者：
vonHippel,PH
中科院分区：
文献类型：
--
作者：
Walstrom,KM;Dozono,JM;Robic,S;vonHippel,PH
本文分析了埃氏菌转录终止因子rho的atp依赖性RNA - DNA解旋酶活性的动力学。利用e的trp t’RNA转录本中的255 nt和391 nt RNA序列组装解旋酶底物。杆菌。这些RNA序列每个都在靠近5 ‘端的位置携带一个rho“装载位点”，在3 ’端的位置携带一个rho依赖的终止序列，互补的~ 20 nt DNA寡核苷酸已被退火。在低盐浓度（50 mM KCl）下进行的反应中，观察到解旋酶活性（DNA低聚物释放）的快速（~ 30 s）预稳态爆发，随后是缓慢的线性阶段。使用poly（rC）或poly（dC）作为一轮活性后释放的rho的陷阱，我们已经证明反应的第一个（爆发）阶段代表了预结合rho六聚体从rho装载位点到RNA分子的3 '端的过程易位。反应的缓慢阶段是复杂的，代表了许多不同过程的组合，包括从rho缓慢释放RNA，将互补的DNA寡核苷酸重新退火到RNA底物，以及将rho六聚体再循环到其他RNA分子上。在较高的盐浓度（150 mM KCl）下进行的反应只包括一个相，因为在这些条件下rho与RNA的解离更快，其振幅对应于每个rho六聚体去除几个DNA低聚物。因此，rho可以在类似于细胞中发现的反应条件下循环并作为催化解旋酶起作用。
The kinetics of the ATP-dependent RNA−DNA helicase activity ofEscherichiacolitranscription termination factor rho have been analyzed. Helicase substrates were assembled using 255 nt and 391 nt RNA sequences from thetrp t‘ RNA transcript ofE. coli. These RNA sequences each carry a rho “loading site” at a position near the 5‘-end, and a rho-dependent terminator sequence at the 3‘-end to which complementary ∼20 nt DNA oligonucleotides have been annealed. A rapid (∼ 30 s) pre-steady-state burst of helicase activity (DNA oligomer release), followed by a slow linear phase, is observed in reactions carried out at low salt concentrations (50 mM KCl). Using poly(rC) or poly(dC) as traps for the rho that is released after one round of activity, we have shown that the first (burst) phase of the reaction represents the processive translocation of prebound rho hexamers from the rho loading site to the 3‘-end of the RNA molecule. The slow phase of the reaction is complex and represents a combination of many different processes, including the slow release of RNA from rho, the reannealing of complementary DNA oligonucleotides to the RNA substrate, and the recycling of rho hexamers onto additional RNA molecules. Reactions carried out at higher salt concentrations (150 mM KCl) consist of only one phase, since under these conditions rho dissociates more rapidly from the RNA, with an amplitude corresponding to several DNA oligomers removed per rho hexamer. Thus, rho can recycle and function as a catalytic helicase under reaction conditions resembling those found in the cell.