Olive J E, Collins R A
Department of Molecular and Medical Genetics, University of Toronto, Toronto, Ontario, Canada M5S 1A8.
Biochemistry. 1998 May 5;37(18):6476-84. doi: 10.1021/bi972584w.
In keeping with the known role of polyamines as counterions in RNA folding, we have found that concentrations of spermine as low as 1 microM facilitate first-order Cis cleavage and decrease the concentration of magnesium required for optimal cleavage of the VS ribozyme. Surprisingly, under certain experimental conditions, cleavage reactions at concentrations of spermine above about 20 microM were not first-order. At 100 microM spermine, about half of the RNA cleaved in an initial very fast burst, k >/= 5 min-1, about 100-fold faster than under our previously optimized conditions; the remainder of the RNA cleaved more slowly. The extent of the burst and the initial rate of cleavage were proportional to RNA concentration, suggesting that the fast phase was due to intermolecular trans cleavage involving two or more RNAs. This inference of trans cleavage was confirmed by demonstrating spermine-dependent trans cleavage of particular combinations of mutant RNAs that were each incapable of cis cleavage. The experimental conditions required to switch the VS ribozyme into trans cleavage mode are quite stringent. The RNA must be preincubated with an adequate concentration of spermine at very low ionic strength near neutral pH. Concentrations of buffers and salts typically used in in vitro studies of ribozymes, including those used in our previous characterization of the VS ribozyme, are sufficiently high that they prevent or reverse the trans cleaving RNA conformation. The ability to switch cleavage modes from cis to trans provides an experimental system to study different active conformations of VS RNA, as well as to investigate the functional consequences of polyamine-RNA interactions.
鉴于多胺作为抗衡离子在RNA折叠中的已知作用，我们发现低至1微摩尔的精胺浓度就能促进一级顺式切割，并降低VS核酶最佳切割所需的镁离子浓度。令人惊讶的是，在某些实验条件下，精胺浓度高于约20微摩尔时的切割反应并非一级反应。在100微摩尔精胺浓度下，约一半的RNA在最初非常快速的爆发中被切割，k≥5分钟-1，比我们之前优化的条件快约100倍；其余的RNA切割得较慢。爆发的程度和初始切割速率与RNA浓度成正比，这表明快速阶段是由于涉及两个或更多RNA的分子间反式切割。通过证明特定组合的突变RNA（每个都不能进行顺式切割）的精胺依赖性反式切割，证实了这种反式切割的推断。将VS核酶转换为反式切割模式所需的实验条件非常严格。RNA必须在接近中性pH的极低离子强度下与足够浓度的精胺预孵育。核酶体外研究中通常使用的缓冲液和盐的浓度，包括我们之前对VS核酶进行表征时使用的那些浓度，都足够高，以至于它们会阻止或逆转反式切割的RNA构象。从顺式到反式切割模式的转换能力提供了一个实验系统，用于研究VS RNA的不同活性构象，以及研究多胺与RNA相互作用的功能后果。
