Complex Formation between a Putative 66-Residue Thumb Domain of Bacterial Reverse Transcriptase RT-Ec86 and the Primer Recognition RNA*
Complex Formation between a Putative 66-Residue Thumb Domain of Bacterial Reverse Transcriptase RT-Ec86 and the Primer Recognition RNA*
复制标题
DOI：
10.1074/jbc.m408462200
复制
发表时间：
2004-12
影响因子：
4.8
通讯作者：
M. Inouye;Haiping Ke;Ariko Yashio;K. Yamanaka;H. Nariya;T. Shimamoto;S. Inouye
中科院分区：
文献类型：
--
作者：
M. Inouye;Haiping Ke;Ariko Yashio;K. Yamanaka;H. Nariya;T. Shimamoto;S. Inouye
逆转录酶（RT）存在于大肠杆菌的少数群体中，负责多拷贝单链DNA的合成。这些RT特异性识别其各自引物-模板RNA中的RNA结构，以从特定内部G残基（分支G残基）的2′-OH基团启动cDNA合成。在此，我们从大肠杆菌中纯化了66个残基的RT-Ec 86，RT的C-末端片段。大肠杆菌，负责多拷贝单链DNA-Ec 86的合成。该片段RT-Ec 86-（255-320）的CD光谱表明主要由α-螺旋结构组成，RT-Ec 86-（255-320）能够结合由5′-寡核苷酸组成的28个碱基的合成RNA。在RT-Ec 86引物-模板RNA体系中，扩增出一条含有分支G残基和识别茎环结构的末端单链RNA，其Kd值为5 × 10-8 m。RT-Ec 86-（255-320）通过逐步缩短RNA的5′端单链区域，仍能形成稳定的复合物，只有由8 bp茎和3个碱基环组成的茎环结构。在这种茎环结构中，UUU环对于复合物的形成是必不可少的。RT-Ec73-（251-316）。大肠杆菌RT不能与RT-Ec 86的28个碱基的RNA结合，但可以与其自身的具有3个碱基的AGU环的茎环结构结合。这些结果支持的概念，细菌RT的高度多样的C-末端区域在识别其自身的特异性引物模板RNA结构的cDNA引发反应中发挥重要作用。
Reverse transcriptases (RT) are found in a minor population of Escherichia coli and are responsible for the synthesis of multicopy single-stranded DNA. These RTs specifically recognize RNA structures in their individual primer-template RNAs to initiate cDNA synthesis from the 2′-OH group of a specific internal G residue (branching G residue). Here, we purified the 66-residue, C-terminal fragment of RT-Ec86, RT from E. coli, which is responsible for the synthesis of multicopy single-stranded DNA-Ec86. This fragment, RT-Ec86-(255–320), was found to consist mainly of α-helical structures on the basis of its CD spectrum, which is consistent with the prediction of this region as the thumb domain from the structural alignment of RT-Ec86 with human immunodeficiency virus-1 RT. RT-Ec86-(255–320) was able to bind to a 28-base synthetic RNA consisting of the 5′-end single-stranded RNA containing the branching G residue and the recognition stem-loop structure in the RT-Ec86 primer-template RNA with a Kd value of 5 × 10–8 m. By stepwise shortening of the 5′-end single-stranded region of the RNA, RT-Ec86-(255–320) was found still to be able to form a stable complex with only the stem-loop structure consisting of an 8-bp stem and a 3-base loop. In this stem-loop structure, the UUU loop was essential for the complex formation. RT-Ec73-(251–316) from another E. coli RT could not bind to the 28-base RNA for RT-Ec86 but could bind to its own stem-loop structure having a 3-base AGU loop. These results support the notion that the highly diverse C-terminal regions of bacterial RTs play an important role in recognizing their own specific primer-template RNA structure for the cDNA priming reaction.