A mechanistic framework for co-transcriptional folding of the HDV genomic ribozyme in the presence of downstream sequence
A mechanistic framework for co-transcriptional folding of the HDV genomic ribozyme in the presence of downstream sequence
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DOI：
10.1016/s0022-2836(02)01027-6
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发表时间：
2002-11-15
影响因子：
5.6
通讯作者：
Bevilacqua, PC
中科院分区：
文献类型：
--
作者：
Diegelman-Parente, A;Bevilacqua, PC
丁型肝炎病毒（HDV）是一种环状致病RNA，通过密切相关的84nt基因组和抗基因组核酶进行自裂解，促进其基因组的复制。每一种核酶的下游是一段被称为衰减子的核苷酸，其作用是与核酶进行碱基对并将其展开成棒状折叠。因此，RNA合成、核酶折叠和切割以及杆折叠的竞争速率可能会影响共转录自切割的效率。在这些研究中，通过监测核酶下游序列长度可变的转录物的共转录自裂，在体外检测基因组核酶的共转录折叠。只有在转录过程中填充非裂解通道的动力学模型才能成功地模拟共转录裂解数据。进入和逃离这些通道的部分影响因素是，可用的衰减序列能否与核酶形成结构，以及这种结构的稳定性。令人惊讶的是，只需要23nt的衰减剂就可以实现强烈的裂解失活。某些3'-含病毒序列的自裂可以通过再生部分恢复；然而，全长衰减物的转录本不能通过体外再生有效地恢复自裂。这表明，在衰减剂存在的情况下，裂解活性核酶折叠不是热力学上最稳定的物种。根据该模型，核酶的自裂效率随着转录速率的降低而提高，然后是全长衰减器。这些结果表明，在没有其他因素的情况下，全长HDV基因组RNA的有效共转录切割可能需要在合成衰减子之前进行切割。(C) 2002 Elsevier Science Ltd.版权所有。
Hepatitis delta virus (HDV) is a circular pathogenic RNA that uses self-cleavage by closely related 84 nt genomic and antigenomic ribozymes to facilitate the replication of its genome. Downstream of each ribozyme is a stretch of nucleotides termed the attenuator that functions to base-pair with and unfold the ribozyme into a rod-like fold. The competing rates of RNA synthesis, ribozyme folding and cleavage, and rod folding are therefore likely to affect the efficiency of co-transcriptional self-cleavage. In these studies, co-transcriptional folding of the genomic ribozyme was assayed in vitro by monitoring co-transcriptional self-cleavage of transcripts having variable lengths of sequence downstream of the ribozyme. Co-transcriptional cleavage data were simulated successfully only with kinetic models in which cleavage-inactive channels were populated during transcription. Partitioning to and escape from these channels was influenced, in part, by whether the available attenuator sequence could form structures with the ribozyme, and by the stability of such structures. Surprisingly, only 23 nt of attenuator were needed for strong inactivation of cleavage. Self-cleavage of certain 3'-virus-containing sequences could be restored, partially, by renaturation; however, self-cleavage of transcripts with a full-length attenuator could not be restored efficiently by renaturation in vitro. This suggests that in the presence of the attenuator, the cleavage-active ribozyme fold is not the thermodynamically most stable species. In accordance with this model, the efficiency of self-cleavage of the ribozyme followed by a full-length attenuator was increased by decreasing the rate of transcription. These results suggest that, in the absence of additional factors, efficient co-transcriptional cleavage of the full-length genomic HDV RNA may require cleavage to occur prior to synthesis of the attenuator. (C) 2002 Elsevier Science Ltd. All rights reserved.