Selective infidelity in diversity-generating retroelements

多样性生成逆转录因子（DGR）是一种独特的、无与伦比的大规模蛋白质生成因子 序列多样性这些元素普遍存在于微生物的“暗物质”中，它们似乎构成了 病毒是微生物生命的主要部分，并且广泛存在于人类病毒组和微生物组中。唯一的其他 在自然界中，脊椎动物的适应性免疫发生了大量蛋白质序列变异， 系统，其中变化使识别新的目标和随之而来的适应动态 环境. DGR似乎也提供了类似的好处。DGRs通过一种 与脊椎动物的免疫系统有着根本不同的机制。在DGR中，多样化源于 遗传信息的传递对一种特定的碱基腺嘌呤是不忠实的，而对其他碱基是忠实的。 这发生在遗传信息从RNA到cDNA的逆转录过程中，并且对 腺嘌呤形成蛋白质功能变异的模式。这种对腺嘌呤的选择性不忠是 DGRs的标志性特征。选择性不忠在生物学中是独一无二的，它是如何发生的尚不清楚。我们取得了 在这个问题上的一个重大突破，通过在体外重建特定的不忠， 博德特氏菌噬菌体DGR。我们已经发现，DGR逆转录酶bRT在与 DGR辅助变异决定簇（Avd）蛋白是合成腺嘌呤-腺嘌呤的必要和充分的蛋白。 诱变的cDNA。我们的结果表明bRT-Avd和DGR RNA联合收割机形成功能性且 结构化核糖核蛋白（RNP）颗粒。我们即将揭开分子和原子 通过以下具体目标详细了解选择性不忠的基础：（1）可视化bRT-Avd/RNA 通过冷冻电子显微镜观察不同功能状态的复合物;（2）鉴定核碱基和 负责选择性不忠的蛋白质决定因素;（3）确定位置的基础- 对特定不忠的特定调制。我们提出的研究将提供根本性的进展， 了解DGR。此外，由于选择性不忠的独特性，这些研究也将 可能提供对控制其它逆转录酶中保真度的机制的新见解（例如，HIV RT） 和核苷酸聚合酶。

Diversity-generating retroelements (DGRs) are unique and unparalleled generators of massive protein sequence diversity. These elements are prevalent in the microbial ‘dark matter’, which appear to comprise a major fraction of microbial life, and are widespread in the human virome and microbiome. The only other example in the natural world of massive protein sequence variation occurs in the vertebrate adaptive immune system, in which variation enables the recognition of novel targets and consequent adaptation to dynamic environments. A similar benefit appears to be provided by DGRs. DGRs diversify proteins through a fundamentally different mechanism than the vertebrate immune system. In DGRs, diversification arises from genetic information being transmitted unfaithfully for one specific base, adenine, and faithfully for the others. This occurs during reverse transcription of genetic information from RNA to cDNA, and the specificity to adenine shapes the pattern of protein functional variation. This selective infidelity to adenines is the central hallmark feature of DGRs. Selectivity infidelity is unique in biology and how it occurs unknown. We have made a significant breakthrough on this problem by reconstituting specific infidelity in vitro for the prototypical Bordetella bacteriophage DGR. We have found that the DGR reverse transcriptase bRT in complex with the DGR accessory variability determinant (Avd) protein is necessary and sufficient to synthesize adenine- mutagenized cDNA. Our results indicate that bRT-Avd and the DGR RNA combine to form a functional and structured ribonucleoprotein (RNP) particle. We are on the verge of uncovering the molecular and atomic details underlying selective infidelity through the following specific aims: (1) Visualize bRT-Avd/RNA complexes in different functional states by cryo-electron microscopy; (2) Identify the nucleobase and protein determinants responsible for selective infidelity; and (3) Determine the basis for position- specific modulation of specific infidelity. Our proposed studies will provide fundamental advances in understanding DGRs. Additionally, because of the uniqueness of selective infidelity, these studies will also likely provide novel insights into mechanisms that control fidelity in other reverse transcriptases (e.g., HIV RT) and nucleotide polymerases in general.