Activation of HIV-specific ribozyme activity by self-cleavage.
Activation of HIV-specific ribozyme activity by self-cleavage.
复制标题
通过自裂解激活 HIV 特异性核酶活性。
DOI：
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发表时间：
1993
影响因子：
14.9
通讯作者：
S. Saragosti
中科院分区：
文献类型：
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作者：
M. Ventura;P. Wang;T. Ragot;M. Perricaudet;S. Saragosti
将设计用于在反式R区切割HIV-1 RNA的锤头核酶插入真核表达载体中。利用位于真核启动子上游的T3 RNA聚合酶启动子对该核酶进行了体外研究。该核酶在任何条件下对其特定靶序列均无活性。为了减少潜在的顺式抑制序列对这种核酶转录物的影响，在核酶编码序列的上游插入了一个特定的靶序列。这种插入允许顺式切割释放具有核酶活性的短RNA，并能够反式切割外部RNA靶标。顺式裂解反应产生两个RNA分子，其中包含催化结构域的较短RNA属反式裂解反应。这种可自切割的核酶在体外37℃下对共享特定靶序列的三种不同的HIV-1转录本具有活性。因此，核酶活性是通过从较长的载体转录物中自切割含有核酶的序列而获得的。
A hammerhead ribozyme designed to cleave in trans the R region of HIV-1 RNA was inserted into a eukaryotic expression vector. This ribozyme was studied in vitro using the T3 RNA polymerase promoter located upstream of the eukaryotic promoter. The ribozyme showed no activity against its specific target sequence under any condition tested. To decrease the influence of potential cis inhibitory sequences in such a ribozyme transcript, a specific target sequence was inserted upstream of the ribozyme-coding sequence. This insertion allowed the release by cis cleavage of a short RNA bearing ribozyme activity and able to cleave in trans an external RNA target. The cis cleavage reaction generated two RNA molecules: the shorter RNA species, which included the catalytic domain, showed a trans cleavage reaction. This self-cleavable ribozyme was active in vitro at 37 degrees C against three distinct HIV-1 transcripts sharing the specific target sequence. Ribozyme activity was thus attained by self-cleavage of the ribozyme-containing sequence from the longer vector transcript.