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首页> 外文期刊>Journal of Molecular Biology >A pH-sensitive RNA tertiary interaction affects self-cleavage activity ofthe HDV ribozymes in the absence of added divalent metal ion
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A pH-sensitive RNA tertiary interaction affects self-cleavage activity ofthe HDV ribozymes in the absence of added divalent metal ion

机译：在不添加二价金属离子的情况下，pH敏感的RNA三级相互作用会影响HDV核酶的自裂解活性

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摘要

Self-cleavage of the genomic and antigenomic ribozymes from hepatitis delta virus (HDV) requires divalent cation for optimal activity. Recently, the HDV genomic ribozyme has been shown to be active in NaCl in the absence of added divalent metal ion at low pH (apparent pK(a) 5.7). However, we find that the antigenomic ribozyme is 100 to 1000-fold less active under similar conditions. With deletion of a three-nucleotide sequence (C41-A42-A43) unique to the genomic ribozyme, the rate constant for cleavage decreased substantially, while activity of the antigenomic ribozyme was enhanced by introducing a CAA sequence. From the crystal structure, it has been proposed that C41 in this sequence is protonated. To investigate a possible connection between activity at low pH and protonation of C41, mutations were made that were predicted to either eliminate protonation or alter the nature of the tertiary interaction upon protonation. In the absence of added Mg2+, these mutations reduced activity and eliminated the observed pH-rate dependence. Thermal denaturation studies revealed a pH-sensitive structural feature in the genomic ribozyme, while unfolding of the mutant ribozymes was pH-independent. We propose that, in the absence of added Mg2+, protonation of C41 contributes to enhanced activity of the HDV genomic ribozyme at low pH.
机译：从肝炎三角洲病毒（HDV）中自我切割基因组和反基因组核酶需要二价阳离子才能实现最佳活性。最近，已证明在低pH下，在不添加二价金属离子的情况下，HDV基因组核酶在NaCl中具有活性（表观pK（a）5.7）。但是，我们发现反基因组核酶在类似条件下的活性降低了100到1000倍。随着基因组核酶特有的三核苷酸序列（C41-A42-A43）的缺失，切割的速率常数显着降低，而反基因组核酶的活性通过引入CAA序列而增强。从晶体结构，已经提出该序列中的C41被质子化。为了研究低pH值活性与C41质子化之间的可能联系，进行了预测可以消除质子化或改变质子化后第三级相互作用性质的突变。在不添加Mg2 +的情况下，这些突变会降低活性并消除观察到的pH速率依赖性。热变性研究揭示了基因组核酶中的pH敏感结构特征，而突变核酶的展开与pH无关。我们建议，在不添加Mg2 +的情况下，C41的质子化有助于在低pH下增强HDV基因组核酶的活性。

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