Highly specific recognition of primer RNA structures for 2′-OH priming reaction by bacterial reverse transcriptases

作     者：Inouye, S Hsu, MY Xu, AG Inouye, M 

作者机构：Robert Wood Johnson Med Sch Dept Biochem Piscataway NJ 08854 USA 

出 版 物：《JOURNAL OF BIOLOGICAL CHEMISTRY》 (生物化学杂志)

年 卷 期：1999年第274卷第44期

页      面：31236-31244页

核心收录：

学科分类：0710[理学-生物学] 071010[理学-生物化学与分子生物学] 081704[工学-应用化学] 07[理学] 08[工学] 0817[工学-化学工程与技术] 

基　　金：NIGMS NIH HHS [GM 44012] Funding Source: Medline 

主　　题：氨基酸序列 碱基序列 DNA 单链 定向分子进化 大肠杆菌/酶学 HIV逆转录酶/遗传学 HIV逆转录酶/代谢 配体 分子序列数据 核酸构象 蛋白质结合 RNA/化学 RNA/代谢 RNA 细菌/化学 RNA 细菌/代谢 RNA指导的DNA聚合酶/遗传学 RNA指导的DNA聚合酶/代谢 重组融合蛋白质类/代谢 反转录因子 底物特异性 

摘      要：A minor population of Escherichia coli contains retroelements called retrons, which encode reverse-transcriptases (RT) to synthesize peculiar satellite DNAs called multicopy single-stranded DNA (msDNA). These RTs recognize specific 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). The resulting products (msDNA) consist of RNA and single-stranded DNA, sharing hardly any sequence homology. Here, we investigated how RT-Ec86 recognizes the specific RNA structure in its primer-template RNA. On the basis of structural comparison with HIV-1 RT, domain exchanges were carried out between two E. coli RTs, RT-Ec86 and RT-Ec73. RT-Ec86 (320 residues) and RT-Ec78 (316 residues) share only 71 identical residues (22%), From the analysis of 10 such constructs, the C-terminal 91-residue sequence of RT-Ec86 was found to be essential for the recognition of the unique stem-loop structure and the branching G residue in the primer-template RNA for retron-Ec86. Using the SELEX (systematic evolution of ligands by exponential enrichment) method with RT-Ec86 and primer RNAs containing random sequences, the identical stem-loop structure (including the 3-U loop) to that found in the retron-Ec86 primer-template RNA was enriched. In addition, the highly conserved 4-base sequence (UAGC), including the branching G residue, was also enriched, These results indicate that the highly diverse C-terminal region recognizes specific stem-loop structures and the branching G residue located upstream of the stem-loop structure. The present results with seemingly primitive RNA-dependent DNA polymerases provide insight into the mechanisms for specific protein RNA recognition.