Specificity of the hairpin ribozyme -: Sequence requirements surrounding the cleavage site
Specificity of the hairpin ribozyme -: Sequence requirements surrounding the cleavage site
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DOI：
10.1074/jbc.274.41.29376
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发表时间：
1999-10-08
影响因子：
4.8
通讯作者：
Berzal-Herranz, A
中科院分区：
文献类型：
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作者：
Pérez-Ruiz, M;Barroso-delJesus, A;Berzal-Herranz, A
发夹核酶的底物序列要求已通过突变和体外选择实验部分确定。人们认为最好的靶标是包含切割位点周围的 N 向下箭头 GUC 序列的靶标。与之前未能评估这些核苷酸的所有可能组合的研究相比，我们对 64 个底物变体的切割进行了详尽的分析，它们代表了 J2/1 核苷酸的所有可能的序列组合，除了已确定的 G(+1) 之外。 24个序列没有观察到切割，C+2变体显示很少或没有切割，而U+2底物都是可切割的，使用AGUC底物获得最大切割率，序列HGUC（H = A、C或U）、GGUN、GGGR（R = A或G）、AGUU的切割率；和 UGUA 比 AGUC 低多达 5 倍。这表明除 NGUC 之外的其他序列也可以被视为良好的目标。第二组序列 WGGG（W = A 或 U）、UGUK（K = G 或 U）、MGAG（M = A 或 C）、AGUA 和 UGGA 的切割效率低 6 到 10 倍。此外，不可切割病毒靶标的 UGCU 序列突变为 AGUC，导致其同源发夹核酶可有效切割底物，这表明我们的结论可以外推到具有不同特异性的其他发夹核酶。
Substrate sequence requirements of the hairpin ribozyme have been partially defined by both mutational and in vitro selection experiments, It was considered that the best targets were those that included the N down arrow GUC sequence surrounding the cleavage site. In contrast to previous studies that failed to evaluate all possible combinations of these nucleotides, we have performed an exhaustive analysis of the cleavage of 64 substrate variants, They represent all possible sequence combinations of the J2/1 nucleotides except the well established G(+1). No cleavage was observed with 24 sequences, C+2 variants showed little or no cleavage, whereas U+2 substrates were all cleavable, The maximal cleavage rate was obtained with the AGUC substrate, Cleavage rates of sequences HGUC (H = A, C, or U), GGUN, GGGR (R = A or G), AGUU; and UGUA were up to 5 times lower than the AGUC one. This shows that other sequences besides NGUC could also be considered as good targets. A second group of sequences WGGG (W = A or U), UGUK (K = G or U), MGAG (M = A or C), AGUA, and UGGA were cleaved between 6 and 10 times less efficiently. Furthermore, the UGCU sequence of a noncleavable viral target was mutated to AGUC resulting in a proficiently cleavable substrate by its cognate hairpin ribozyme, This indicates that our conclusions may be extrapolated to other hairpin ribozymes with different specificity.