Nahas Michelle K, Wilson Timothy J, Hohng Sungchul, Jarvie Kaera, Lilley David M J, Ha Taekjip
Physics Department, University of Illinois, Urbana-Champaign, Urbana, Illinois 61801, USA.
Nat Struct Mol Biol. 2004 Nov;11(11):1107-13. doi: 10.1038/nsmb842. Epub 2004 Oct 10.
We have used single-molecule spectroscopy to untangle conformational dynamics and internal chemistry in the hairpin ribozyme. The active site of the ribozyme is stably formed by docking two internal loops, but upon cleavage undocking is accelerated by two orders of magnitude. The markedly different kinetic properties allow us to differentiate cleaved and ligated forms, and thereby observe multiple cycles of internal cleavage and ligation of a ribozyme in a uniquely direct way. The position of the internal equilibrium is biased toward ligation, but the cleaved ribozyme undergoes several undocking events before ligation, during which products may dissociate. Formation of the stably docked active site, rapid undocking after cleavage, and a strong bias toward ligation should combine to generate a stable circular template for the synthesis of the viral (+) strand and thus ensure a productive replication cycle.
我们利用单分子光谱技术来解析发夹状核酶的构象动力学和内部化学过程。核酶的活性位点通过两个内部环的对接稳定形成，但在切割时，解对接加速了两个数量级。显著不同的动力学性质使我们能够区分切割和连接形式，从而以独特的直接方式观察核酶内部切割和连接的多个循环。内部平衡位置偏向连接，但切割后的核酶在连接前会经历几次解对接事件，在此期间产物可能解离。稳定对接的活性位点的形成、切割后快速解对接以及对连接的强烈偏向应共同作用，为病毒（+）链的合成生成一个稳定的环状模板，从而确保一个有效的复制周期。
