Walstrom K M, Dozono J M, Robic S, von Hippel P H
Institute of Molecular Biology, University of Oregon, Eugene, Oregon 97403, USA.
Biochemistry. 1997 Jul 1;36(26):7980-92. doi: 10.1021/bi963179s.
The kinetics of the ATP-dependent RNA-DNA helicase activity of Escherichia colitranscription termination factor rho have been analyzed. Helicase substrates were assembled using 255 nt and 391 nt RNA sequences from the trp t' RNA transcript of E. 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 approximately 20 nt DNA oligonucleotides have been annealed. A rapid ( approximately 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.
已对大肠杆菌转录终止因子rho的ATP依赖性RNA-DNA解旋酶活性的动力学进行了分析。使用来自大肠杆菌trp t' RNA转录本的255 nt和391 nt RNA序列组装解旋酶底物。这些RNA序列在靠近5'端的位置各携带一个rho“加载位点”，在3'端携带一个rho依赖性终止子序列，与之互补的约20 nt DNA寡核苷酸已退火至该序列上。在低盐浓度（50 mM KCl）下进行的反应中，观察到解旋酶活性（DNA寡聚物释放）有一个快速（约30秒）的预稳态爆发，随后是一个缓慢的线性阶段。使用聚（rC）或聚（dC）作为一轮活性后释放的rho的捕获剂，我们已经表明反应的第一（爆发）阶段代表预先结合的rho六聚体从rho加载位点向RNA分子3'端的进行性易位。反应的缓慢阶段很复杂，代表许多不同过程的组合，包括RNA从rho的缓慢释放、互补DNA寡核苷酸与RNA底物的重新退火以及rho六聚体在其他RNA分子上的循环利用。在较高盐浓度（150 mM KCl）下进行的反应仅由一个阶段组成，因为在这些条件下rho从RNA上解离得更快，其幅度对应于每个rho六聚体去除几个DNA寡聚物。因此，在类似于细胞内发现的反应条件下，rho可以循环并作为催化解旋酶发挥作用。
