Department of Molecular Biology & Biochemistry, Simon Fraser University, Burnaby, British Columbia, Canada V5A 1S6.
J Mol Biol. 2010 Jan 15;395(2):234-41. doi: 10.1016/j.jmb.2009.11.028. Epub 2009 Nov 13.
The 8-17 deoxyribozyme is an in vitro selected enzyme capable of sequence-specific cleavage of RNA. While selected to be a magnesium and zinc-utilizing enzyme, the 8-17 DNAzyme has been shown to utilize lead for its catalysis. Fluorescence-based experiments have indicated that the magnesium- and zinc-utilizing versions of the DNAzyme-substrate complex need to form a defined tertiary structure to be active, but no such global folding is required for the lead-mediated activity. Here, we have investigated this phenomenon, including the use of contact photo-crosslinking to map the tertiary fold of the lead-dependent DNAzyme. While our results recapitulate that global folding is not required for the lead activity, they reveal strikingly distinct lead-mediated modes of activity under conditions of low versus moderate solution ionic strength. Even in very low salt buffers, where no global folding of the 8-17 DNAzyme occurs, the active site of the enzyme appears to form a distinct local fold, one that cannot be modelled easily by DNA/RNA constructs that preserve key sequence and secondary structure features of the active site.
8-17 脱氧核酶是一种体外筛选出的酶，能够特异性切割 RNA。虽然被选为利用镁和锌的酶，但 8-17 DNA 酶已被证明利用铅进行催化。基于荧光的实验表明，需要形成特定的三级结构才能使镁和锌利用的 DNA 酶-底物复合物具有活性，但铅介导的活性不需要这种全局折叠。在这里，我们研究了这一现象，包括使用接触光交联来绘制依赖铅的 DNA 酶的三级折叠。虽然我们的结果表明，全局折叠不是铅活性所必需的，但它们揭示了在低离子强度和中等离子强度条件下，铅介导的活性具有截然不同的模式。即使在低盐缓冲液中，8-17 DNA 酶也不会发生全局折叠，酶的活性位点似乎形成了一个独特的局部折叠，这不能通过保留活性位点关键序列和二级结构特征的 DNA/RNA 结构来轻松建模。
