Reverse Transcriptases in the Prokaryotes

描述（由申请人提供）：与早期的信仰相反， 在细菌中也发现了retroelements。Retron是这样的元素， 这些存在于大肠杆菌的一些野生菌株中（<10%的 种群）、黄色粘球菌（所有天然分离株）和霍乱弧菌 (only在致病菌株中）。它们编码逆转录酶（RT） 在进化上与逆转录病毒RT和其他真核RT相关。细菌 RT负责合成一种特殊的卫星单链 称为msDNA（多拷贝单链DNA）的DNA，其中单链DNA cDNA是从高度结构化的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合成的未解之谜是个体如何 彼此高度不同的细菌RT能够识别它们的同源RNA 分子，以及cDNA合成如何从特定的内部G残基引发 （分支G残基）在单个RNA分子。我们最近证明， RT-Ec 86的91个残基的C-末端结构域负责特异性的 识别引物-模板RNA分子特有的茎环结构 对于RT-EC 86。值得注意的是，这种识别茎环结构位于下游 用于cDNA引发反应的分支G残基。的基础上 根据我们最近的研究结果，我们假设看似原始的细菌RT 保留了利用C-末端拇指结构域区域的独特能力， 特异性识别它们的同源茎环结构下游的 分支G残基这种独特的RNA-拇指结构域相互作用允许2 '-OH 将分支G残基的基团正确定位在活性位点 添加了与模板RNA互补的第一个核苷酸的RT 形成2 ′，5 ′-磷酸二酯键。 本研究首先尝试构建RT-Ec 86的分子模型， 其与引物-模板RNA和最终msDNA产物的复合物。上 在这些模型的基础上，我们将采取实验方法来确定 拇指结构域与 识别茎-环结构，用于来自 分支G残基的2’-OH基团，以及用于cDNA延伸反应 产生最终的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.