Selective infidelity in diversity-generating retroelements

多样性生成逆转录元件(DGR)是产生大量蛋白质的独特和无与伦比的生成器 序列分集。这些元素普遍存在于微生物的“暗物质”中，它们似乎包含一种 是微生物生命的主要组成部分，广泛存在于人类病毒体和微生物群中。唯一的另一个 例如自然界中大量蛋白质序列的变异发生在脊椎动物的适应性免疫中 系统，在该系统中，变化使得能够识别新的目标并由此适应动态 环境。DGRs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，多样化产生于 不忠实地为一个特定的碱基，腺嘌呤传递遗传信息，而为其他碱基忠实地传递遗传信息。 这发生在遗传信息从RNA逆转录到cdna的过程中，并且特异性 腺嘌呤塑造了蛋白质功能变异的模式。这种对腺嘌呤的选择性不忠是 DGRS的标志性功能。选择性不忠在生物学中是独一无二的，它是如何发生的还不得而知。我们已经做出了 在这个问题上的重大突破，通过在体外为原型重建特定的不忠 噬菌体DGR。我们已经发现DGR逆转录酶BRT在与 DGR辅助变异性决定簇(Avd)蛋白是合成腺嘌呤的必要条件和充分条件。 诱变的cDNA.我们的结果表明，BRT-Avd和DGR RNA结合形成了功能和 结构化核糖核蛋白(RNP)颗粒。我们即将揭开分子和原子的面纱 通过以下具体目标详细说明选择性不忠的潜在原因：(1)可视化BRT-Avd/RNA 冰冻电子显微镜观察不同功能状态的络合物；(2)鉴定核碱基和 负责选择性不忠的蛋白质决定因素；以及(3)决定位置的基础- 对特定不忠的特定调制。我们建议的研究将在以下方面提供基本的进展 了解DGRS。此外，由于选择性不忠的独特性，这些研究还将 可能为控制其他逆转录酶(如HIV RT)的保真度的机制提供了新的见解 和一般的核苷酸聚合酶。

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.