Lustig B, Lin N H, Smith S M, Jernigan R L, Jeang K T
Laboratory of Mathematical Biology, NCI, NIAID, NIH, Bethesda, MD 20892-0460, USA.
Nucleic Acids Res. 1995 Sep 11;23(17):3531-8. doi: 10.1093/nar/23.17.3531.
A prototypic hammerhead ribozyme has three helices that surround an asymmetrical central core loop. We have mutagenized a hammerhead type ribozyme. In agreement with previous studies, progressive removal of stem-loop II from a three stemmed ribozyme showed that this region is not absolutely critical for catalysis. However, complete elimination of stem II and its loop did reduce, but did not eliminate, function. In a stem-loop II-deleted ribozyme, activity was best preserved when a purine, preferably a G, was present at position 10.1. This G contributed to catalysis irregardless of its role as either one part of a canonical pair with a C residue at 11.1 or a lone nucleotide with C (11.1) deleted. Computational methods using lattices generated 87 million three-dimensional chain forms for a stem-loop II-deleted RNA complex that preserved one potential G.C base pair at positions 10.1 and 11.1. This exhaustive set of chain forms included one major class of structures with G(10.1) being spatially proximal to the GUCX cleavage site of the substrate strand. Strong correlations were observed between colinear arrangement of stems I and III, constraints of base-pairing in the central core loop, and one particular placement of G(10.1) relative to the cleavage site. Our calculations of a stem-loop II-deleted ribozyme indicate that without needing to invoke any other constraints, the inherent asymmetry in the lengths of the two loop strands (3 nt in one and 7 nt in the other) that compose the core and flank G10.1-C11.1 stipulated strongly this particular G placement. This suggests that the hammerhead ribozyme maintains an asymmetry in its internal loop for a necessary structure/function reason.
一个典型的锤头状核酶有三个围绕不对称中央核心环的螺旋。我们对一种锤头状核酶进行了诱变。与先前的研究一致，从三茎核酶中逐步去除茎环II表明该区域对催化并非绝对关键。然而，完全消除茎II及其环确实降低了但并未消除功能。在一个缺失茎环II的核酶中，当在10.1位存在嘌呤，最好是鸟嘌呤时，活性得到了最佳保留。无论该鸟嘌呤作为与11.1位的胞嘧啶残基形成规范碱基对的一部分，还是作为11.1位胞嘧啶缺失后的单个核苷酸，它都对催化有贡献。使用晶格的计算方法为一个缺失茎环II的RNA复合物生成了8700万个三维链形式，该复合物在10.1和11.1位保留了一个潜在的G·C碱基对。这一详尽的链形式集合包括一类主要结构，其中G(10.1)在空间上靠近底物链的GUCX切割位点。观察到茎I和茎III的共线排列、中央核心环中碱基配对的限制以及G(10.1)相对于切割位点的一个特定位置之间存在强相关性。我们对缺失茎环II的核酶的计算表明，无需引入任何其他限制，组成核心并侧翼G10.1 - C11.1的两条环链（一条3个核苷酸，另一条7个核苷酸）长度上的固有不对称性强烈规定了这种特定的鸟嘌呤位置。这表明锤头状核酶出于必要的结构/功能原因在其内部环中保持不对称性。
