Pan T, Artsimovitch I, Fang X W, Landick R, Sosnick T R
Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, IL 60637, USA.
Proc Natl Acad Sci U S A. 1999 Aug 17;96(17):9545-50. doi: 10.1073/pnas.96.17.9545.
We compared in vitro transcription-initiated folding of the ribozyme from Bacillus subtilis RNase P to refolding from the full-length, denatured state by monitoring the appearance of its catalytic activity. At 37 degrees C, Mg(2+)-initiated refolding of the wild type and a circularly permutate ribozyme takes minutes and is limited by a kinetic trap. Transcription by T7 RNA polymerase alters the folding pathway of both RNAs and introduces new kinetic traps. Transcription by the core Escherichia coli RNA polymerase yields the same result, in spite of its 4-fold-slower elongation rate. However, the presence of its elongation factor NusA accelerates more than 10-fold the transcription-initiated folding of the circularly, permutated ribozyme by E. coli RNA polymerase. The effect of NusA likely is caused by its enhancement of transcriptional pausing because NusA did not accelerate transcription-initiated folding using a mutant RNA polymerase that failed to pause or respond to NusA during ribozyme synthesis. We conclude that both transcription and specific pausing therein can alter RNA-folding pathways.
我们通过监测其催化活性的出现，比较了枯草芽孢杆菌核糖核酸酶P核酶的体外转录起始折叠与从全长变性状态的重折叠情况。在37摄氏度时，镁离子引发的野生型和环状排列核酶的重折叠需要数分钟，并且受到动力学陷阱的限制。T7 RNA聚合酶的转录改变了两种RNA的折叠途径，并引入了新的动力学陷阱。尽管核心大肠杆菌RNA聚合酶的延伸速率慢4倍，但其转录产生了相同的结果。然而，其延伸因子NusA的存在使大肠杆菌RNA聚合酶引发的环状排列核酶的转录起始折叠加速了10倍以上。NusA的作用可能是由于其增强了转录暂停，因为在核酶合成过程中，NusA使用无法暂停或对NusA作出反应的突变RNA聚合酶时，并未加速转录起始折叠。我们得出结论，转录及其特定的暂停都可以改变RNA折叠途径。
