Wang L, Ruffner D E
Department of Bioengineering, University of Utah, Salt Lake City, UT 84108, USA.
Nucleic Acids Res. 1997 Nov 1;25(21):4355-61. doi: 10.1093/nar/25.21.4355.
The hammerhead domain is one of the smallest known ribozymes. Like other ribozymes it catalyzes site-specific cleavage of a phosphodiester bond. The hammerhead ribozyme has been the subject of a vast number of biochemical and structural studies aimed at determining the structure and mechanism of cleavage. Recently crystallographic analysis has produced a structure for the hammerhead. As the hammerhead is capable of undergoing cleavage within the crystal, it would appear that the crystal structure is representative of the catalytically active solution structure. However, the crystal structure conflicts with much of the biochemical data and reveals a catalytic metal ion binding site expected to be of very low affinity. Clearly, additional studies are needed to reconcile the discrepancies and provide a clear understanding of the structure and mechanism of the hammerhead ribozyme. Here we demonstrate that a unique crosslink can be induced in the hammerhead with 2-thiocytidine or 4-thiouridine substitution at different locations within the conserved core. Generation of the same crosslink with different modifications at different positions suggests that the structure trapped by the crosslink may be relevant to the catalytically active solution structure of the hammerhead ribozyme. As this crosslink appears to be incompatible with the crystal structure, this provides yet another indication that the active solution and crystal structures may differ significantly.
锤头状结构域是已知最小的核酶之一。与其他核酶一样，它催化磷酸二酯键的位点特异性切割。锤头状核酶一直是大量生物化学和结构研究的对象，这些研究旨在确定切割的结构和机制。最近的晶体学分析得出了锤头状结构的结构。由于锤头状结构能够在晶体内进行切割，因此晶体结构似乎代表了催化活性溶液结构。然而，晶体结构与许多生物化学数据相冲突，并揭示了一个预期亲和力非常低的催化金属离子结合位点。显然，需要进行更多研究来协调这些差异，并清晰地理解锤头状核酶的结构和机制。在这里，我们证明在保守核心内不同位置用2-硫代胞苷或4-硫尿苷取代可在锤头状结构中诱导出独特的交联。在不同位置用不同修饰产生相同的交联表明，被交联捕获的结构可能与锤头状核酶的催化活性溶液结构相关。由于这种交联似乎与晶体结构不相容，这再次表明活性溶液结构和晶体结构可能有显著差异。
