Reverse Transcriptases in the Prokaryotes

描述(由申请人提供)：与早先的信念相反， 在细菌中也发现了逆转录因子，逆转录因子包括 它们存在于一些野生菌株中(10%的 种群)、黄色粘球菌(均为天然分离株)和霍乱弧菌 (仅限于致病菌株)。它们编码逆转录酶(RT) 在进化上与逆转录病毒RTS和其他真核RTS相关。细菌 RTS负责合成一种特殊的单链卫星 DNA称为msDNA(多拷贝单链DNA)，其中单链DNA (CDNA)是从高度结构的RNA的内部Rg残基分支出来的 形成独特的2‘，5’-磷酸二酯键的分子。 关于msDNA合成的悬而未决的谜团是 高度不同的细菌RT能够识别它们的同源RNA 分子，以及DNA合成是如何从特定的内部G残基启动的 (支化G残基)在单个RNA分子中。我们最近证明了 RT-EC86的91个残基的C末端结构域负责特异的 识别引物-模板RNA分子特有的茎环结构 用于RT-EC86。值得注意的是，这种识别茎环结构位于下游 所述支化G残基用于所述cDNA启动反应。在…的基础上 我们最近的结果，我们假设看似原始的细菌RTS 保留利用C-末端拇指区域的独特能力 特别识别它们下游的同源茎环结构 分支G残留物。这种独特的RNA-拇指结构域相互作用允许2‘-OH 将支化G残基的基团正确定位在活性部位 在其上添加与模板RNA互补的第一核苷酸的RTS 形成2‘，5’-磷酸二酯键。 在本方案中，我们首先尝试构建RT-EC86和 它与引物-模板RNA和最终的msDNA产物形成复合体。论 基于这些模型，我们将采取实验方法来确定 拇指指域和拇指之间相互作用的精确分子机制 识别茎环结构，用于来自 支化G残基的2‘-OH基团，以及用于cDNA延伸反应 最终得到msDNA产物。

DESCRIPTION (provided by applicant): Contrary to the earlier belief, retroelements have also been found in bacteria Retrons are such elements as these existing in some wild strains of Escherichia coli (<10 percent of the population), Myxococcus xanthus (all natural isolates), and Vibrio cholereae (only in pathogenic strains). They encode reverse transcriptase (RT) evolutionarily related to retroviral RTs and other eukaryotic RTs. Bacterial RTs are responsible for the synthesis of a peculiar satellite single-stranded DNA called msDNA (multicopy single-stranded DNA), in which a single strand DNA (cDNA) is branched out from an internal rG residue of a highly structured RNA molecule forming a unique 2' ,5 '-phosphodiester linkage. The outstanding mysteries concerning msDNA synthesis are how individual bacterial RTs highly diverse each other are able to recognize their cognate RNA molecule, and how cDNA synthesis is primed from a specific internal G residue (branching G residue) in the single RNA molecule. We recently demonstrated that the 91-residue C-terminal domain of RT-Ec86 is responsible for the specific recognition of a stem-loop structure unique to the primer-template RNA molecule for RT-Ec86. Notably, this recognition stem-loop structure locates downstream of the branching G residue used for the cDNA priming reaction. On the basis of our recent results, we hypothesize that the seemingly primitive bacterial RTs retain the unique ability to utilize the C-terminal thumb domain region to specifically recognize their cognate stem-loop structure downstream of the branching G residue. This unique RNA-thumb domain interaction allows the 2'-OH group of the branching G residue to be correctly positioned at the active site of RTs to which the first nucleotide complimentary to the template RNA is added forming a 2', 5 '-phosphodiester linkage. In this proposal, we first attempt to construct molecular models of RT-Ec86 and its complexes with the primer-template RNA and the final msDNA product. On the basis of these models, we will take experimental approaches to determine the precise molecular mechanisms for the interaction between the thumb domain and the recognition stem-loop structure, for the cDNA priming reaction from the 2'-OH group of the branching G residue, and for the cDNA elongation reaction leading to the final msDNA product.