SELF-CLEAVAGE OF HEPATITIS DELTA-VIRUS GENOMIC STRAND RNA IS ENHANCED UNDER PARTIALLY DENATURING CONDITIONS
SELF-CLEAVAGE OF HEPATITIS DELTA-VIRUS GENOMIC STRAND RNA IS ENHANCED UNDER PARTIALLY DENATURING CONDITIONS
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DOI：
10.1021/bi00487a002
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发表时间：
1990-09-04
期刊：
影响因子：
2.9
通讯作者：
BEEN, MD
中科院分区：
文献类型：
--
作者：
ROSENSTEIN, SP;BEEN, MD
含有来自丁型肝炎病毒基因组链的自动切割序列的多核糖核苷酸的自切割因RNA结构不稳定的条件而增强。本研究中使用的转录本的自裂解需要 Mg2+（或另一种二价阳离子），并且在没有变性剂的情况下，在非常低的 Mg2+ 浓度（0.05-0.1 mM）下观察到最大裂解。然而，在37度。 C 和在 2-10 mM Mg2+ 存在的情况下，当添加尿素至 5 M 或甲酰胺至 10 M 时，裂解速率增加了 50 倍。较高浓度的相同试剂（9.5 M 尿素或 22.5 M 甲酰胺）可阻止裂解，可能是因为自裂解所需的结构被强变性条件破坏。与之前的报告相比 [Wu, H.-N., and Lai, M. M. C. (1989) Science 243, 652-654]，我们发现用 EDTA 螯合 Mg2+ 可以终止裂解反应，但不会促进可测量量的裂解产物连接。变性剂在体外促进快速自裂解的能力增加了一种未识别的因子在体内具有类似作用的可能性。
Self-cleavage of a polyribonucleotide containing an autocleaving sequence from the genomic strand of hepatitis delta virus was enhanced by conditions that destabilized RNA structure. Self-cleavage of the transcripts used in this study required Mg2+ (or another divalent cation), and in the absence of denaturants, maximum cleavage was observed at very low Mg2+ concentrations (0.05-0.1 mM). However, at 37.degree. C and in the presence of 2-10 mM Mg2+ the rate of cleavage was increased as much as 50-fold with the addition of urea to 5 M or formamide to 10 M. Cleavage was prevented by higher concentrations of the same reagents (9.5 M urea or 22.5 M formamide), presumably because a structure required for self-cleavage is disrupted by strongly denaturing conditions. In contrast to a previous report [Wu, H.-N., and Lai, M. M. C. (1989) Science 243, 652-654], we find that chelating Mg2+ with EDTA terminates the cleavage reaction without promoting measurable amounts of ligation of the cleavage products. The ability of denaturants to promote rapid self-cleavage in vitro raises the possibility that an unidentified factor could have a similar effect in vivo.