Expression of an active form of recombinant Ty1 reverse transcriptase in Escherichia coli:: a fusion protein containing the C-terminal region of the Ty1 integrase linked to the reverse transcriptase-RNase H domain exhibits polymerase and RNase H activities
Expression of an active form of recombinant Ty1 reverse transcriptase in Escherichia coli:: a fusion protein containing the C-terminal region of the Ty1 integrase linked to the reverse transcriptase-RNase H domain exhibits polymerase and RNase H activities
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DOI：
10.1042/0264-6021:3480337
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发表时间：
2000-06-01
影响因子：
4.1
通讯作者：
Wilhelm, FX
中科院分区：
文献类型：
--
作者：
Wilhelm, M;Boutabout, M;Wilhelm, FX
酿酒酵母Ty 1逆转录转座子的复制需要能够合成Ty 1 DNA的逆转录酶。本文首次报道了具有聚合酶和RNase H活性的重组Ty 1酶的活性形式。Ty 1酶在大肠杆菌中表达为六组氨酸标记的融合蛋白，以便于通过金属螯合层析纯化重组蛋白。只有当整合酶基因编码的氨基酸残基被添加到逆转录酶-RNase H结构域的N-末端时，才检测到重组蛋白的催化活性。这表明整合酶结构域可能在逆转录酶的正确折叠中起作用。分析了Ty 1酶的几种生化性质，包括MgCl 2、NaCl、温度和链终止剂双脱氧GTP对其聚合酶活性的影响。通过监测RNA-DNA模板-引物的切割来检查RNA酶H活性。我们的研究结果表明，RNase H和聚合酶活性位点之间的距离对应于14个核苷酸的RNA-DNA异源双链体的长度。在E.大肠杆菌中的整合酶，将有助于进一步的生化和结构分析，并为更好地了解整合酶的作用，在激活的逆转录酶。
Replication of the Saccharomyces cerevisiae Ty1 retrotransposon requires a reverse transcriptase capable of synthesizing Ty1 DNA. The first description of an active form of a recombinant Ty1 enzyme with polymerase and RNase H activities is reported here. The Ty1 enzyme was expressed as a hexahistidine-tagged fusion protein in Escherichia coli to facilitate purification of the recombinant protein by metal-chelate chromatography. Catalytic activity of the recombinant protein was detected only when amino acid residues encoded by the integrase gene were added to the N-terminus of the reverse transcriptase-RNase H domain. This suggests that the integrase domain could play a role in proper folding of reverse transcriptase, Several biochemical properties of the Ty1 enzyme were analysed, including the effect of MgCl2, NaCl, temperature and of the chain terminator dideoxy GTP on its polymerase activity. RNase H activity was examined by monitoring the cleavage of a RNA-DNA template-primer. Our results suggest that the distance between the RNase H and polymerase active sites corresponds to the length of a 14-nucleotide RNA-DNA heteroduplex. The recombinant protein produced in E. coli should be useful for further biochemical and structural analyses and for a better understanding of the role of integrase in the activation of reverse transcriptase.