Selective infidelity in diversity-generating retroelements

产生多样性的逆向因素中的选择性不忠

• 批准号: 10305645
• 负责人: PARTHO GHOSH
• 金额: $ 31.6万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-12-01 至 2023-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10305645
• 关键词: Adaptive Immune System; Adenine; Amino Acid Sequence; Amino Acids; Bacteriophages; Biology; Bordetella; Complementary DNA; Complex; Cryoelectron Microscopy; Crystallization; Elements; Environment; Frequencies; Goals; Grain; Group Structure; HIV; Higher Order Chromatin Structure; Human Microbiome; Immune system; In Vitro; Introns; Knowledge; Life; Maps; Modeling; Molecular; Mutate; Mutation; Nucleotides; Pattern; Polymerase; Positioning Attribute; Proteins; RNA; RNA-Directed DNA Polymerase; Resolution; Retroelements; Retroviridae; Reverse Transcription; Ribonucleoproteins; Role; Shapes; Site; Specificity; Structure; Techniques; Testing; Variant; Visualization; Work; analog; base; dark matter; genetic element; genetic information; human virome; in silico; in vivo; insight; microbial; novel; nucleobase; nucleobase analog; particle; reconstitution

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.

多样性生成逆转录元件 (DGR) 是独特且无与伦比的大量蛋白质生成器 这些元素在微生物“暗物质”中普遍存在，它们似乎包含 微生物生命的主要部分，广泛存在于人类病毒组和微生物组中。 自然界中脊椎动物适应性免疫中发生大量蛋白质序列变异的例子 系统，其中变化能够识别新目标并随后适应动态 DGR 似乎通过使蛋白质多样化提供了类似的好处。 与脊椎动物免疫系统有着根本不同的机制。在 DGR 中，多样化源自于免疫系统。 遗传信息对于一种特定碱基（腺嘌呤）不忠实地传递，而对于其他碱基则忠实地传递。 这种情况发生在遗传信息从 RNA 逆转录为 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核心。 DGR 的标志性特征是生物学上的独特性，但我们仍不清楚它是如何发生的。 通过在体外重构原型的特定不忠行为，在这个问题上取得了重大突破 我们发现博德特氏菌噬菌体 DGR 逆转录酶 bRT 与 BRT 形成复合物。 DGR 辅助变异决定子 (Avd) 蛋白对于合成腺嘌呤是必要且充分的 我们的结果表明 bRT-Avd 和 DGR RNA 结合形成功能性和 我们即将揭开分子和原子的神秘面纱。 通过以下具体目标详细说明选择性不忠的背后：(1) 可视化 bRT-Avd/RNA 通过冷冻电子显微镜鉴定不同功能状态的复合物；（2）识别核碱基和 导致选择性不忠的蛋白质决定因素；以及（3）确定位置的基础 我们提出的研究将为特定不忠的特定调节提供根本性进展。 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（例如 HIV RT）保真度的机制提供新的见解 和一般的核苷酸聚合酶。