A role for upstream RNA structure in facilitating the catalytic fold of the genomic hepatitis delta virus ribozyme
A role for upstream RNA structure in facilitating the catalytic fold of the genomic hepatitis delta virus ribozyme
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DOI：
10.1006/jmbi.2000.3953
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发表时间：
2000-08-11
影响因子：
5.6
通讯作者：
Bevilacqua, PC
中科院分区：
文献类型：
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作者：
Chadalavada, DM;Knudsen, SM;Bevilacqua, PC
丁型肝炎病毒(HDV)具有环状的RNA基因组，通过双滚环机制进行复制。HDV的基因组和抗基因组版本包含一个核酶，它经历顺式切割，从而将转录物加工成单位长度的单体。一个包含30个核苷酸的HDV基因组转录本被发现在裂解位点的上游减弱了自我裂解。结构作图和定点突变揭示了由上游核苷酸-24至-15组成的抑制延伸，形成了称为Alt 1的长距离配对，P2的3‘链(P2(3’))位于核酶的3‘端。发现了另外两个可供选择的配对，Alt 2，涉及上游核苷酸-核酶相互作用，以及Alt 3，涉及核酶-核酶相互作用。通过加入DNA寡聚体，自切割被拯救了2700到20,000倍，它隔离了反式中的-24/-15抑制伸展。令人惊讶的是，当上游核苷酸的数量增加到54时，共转录的自我切割就会发生。计算机预测和结构图谱支持存在一个异常稳定的上游发夹，涉及核苷酸-54到-18，称为P(-1)/L(-1)，它隔离了顺式基因-24/-15抑制伸展的大部分。这个发夹之后是一段单链的富含嘧啶的核苷酸，称为J(-1/1)。序列比较表明，P(-1)/L(-1)/J(-1/1)基序在已知的基因组HDV分离株中保守，J(-1/1)拉伸在抗基因组HDV分离株中保守。最后，含有Alt1的核酶的二级结构为了解核酶可能的折叠中间体提供了线索。(C)2000年学术出版社。
Hepatitis delta virus (HDV) has a circular RNA genome that replicates by a double rolling-circle mechanism. The genomic and antigenomic versions of HDV contain a ribozyme that undergoes cis-cleavage, thereby processing the transcript into unit-length monomers. A genomic HDV transcript containing 30 nucleotides immediately upstream of the cleavage site was found to have attenuated self-cleavage. Structure mapping and site-directed mutagenesis revealed an inhibitory stretch consisting of upstream nucleotides -24 to -15 that forms a long-range pairing, termed Alt 1, with the 3' strand of P2 (P2(3')) located at the very 3'-end of the ribozyme. Two other alternative pairings were found, Alt 2, which involves upstream nucleotide-ribozyme interactions, and Alt 3, which involves ribozyme-ribozyme interactions. Self-cleavage was rescued 2700 to 20,000-fold by adding DNA oligomers, which sequester the -24/-15 inhibitory stretch in trans. Surprisingly, co-transcriptional self-cleavage occurs when the number of upstream nucleotides is increased to 54. Computer prediction and structure mapping support the existence of an unusually stable upstream hairpin involving nucleotides -54 to -18, termed P(-1)/L(-1), which sequesters the majority of the -24/-15 inhibitory stretch in cis. This hairpin is followed by a stretch of single-stranded pyrimidine-rich nucleotides, termed J(-1/1). Sequence comparison suggests that the P(-1)/L(-1)/J(-1/1) motif is conserved among known genomic HDV isolates, and that the J(-1/1) stretch is conserved among antigenomic HDV isolates. Lastly, the secondary structure of the Alt 1-containing ribozyme provides insight into possible folding intermediates of the ribozyme. (C) 2000 Academic Press.