Guo H C, Collins R A
Canadian Institute for Advanced Research Program in Evolutionary Biology, University of Toronto, Ontario.
EMBO J. 1995 Jan 16;14(2):368-76. doi: 10.1002/j.1460-2075.1995.tb07011.x.
We have constructed a ribozyme containing 144 nucleotides of Neurospora VS RNA that can catalyze the cleavage of a separate RNA in a true enzymatic manner (Km approximately 0.13 microM, kcat approximately 0.7/min). Comparison of the rates of cis- and trans-cleavage, as well as the lack of effect of pH on the rate of cleavage, suggest that a rate-limiting step, possibly a conformational change, occurs prior to cleavage. The minimum contiguous substrate sequence required for cleavage consists of one nucleotide upstream and 19 nucleotides downstream of the cleavage site. Unlike most other ribozymes which interact with long single-stranded regions of their substrates, the minimal substrate for the VS ribozyme consists mostly of a stable stem-loop, which would appear to preclude its recognition simply via extensive Watson-Crick base pairing.
我们构建了一种包含144个核苷酸的脉孢菌VS RNA的核酶，它能够以真正的酶促方式催化另一种RNA的切割（米氏常数约为0.13微摩尔，催化常数约为0.7/分钟）。顺式和反式切割速率的比较，以及pH对切割速率缺乏影响，表明在切割之前发生了一个限速步骤，可能是构象变化。切割所需的最小连续底物序列由切割位点上游的一个核苷酸和下游的19个核苷酸组成。与大多数其他与底物的长单链区域相互作用的核酶不同，VS核酶的最小底物主要由一个稳定的茎环组成，这似乎排除了仅通过广泛的沃森-克里克碱基配对进行识别的可能性。
