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: 9896180
• 负责人: PARTHO GHOSH
• 金额: $ 31.5万
• 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/9896180
• 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.

产生多样性的追溯元件（DGRS）是巨大蛋白质的独特且无与伦比的发电机 序列多样性。这些元素在微生物“暗物质”中很普遍，这似乎建立了一个 微生物寿命的主要部分，并且在人类病毒组和微生物组中广泛。唯一的 大规模蛋白质序列变化的自然世界中发生在脊椎动物适应性免疫中 系统，其中变异能够识别新目标，并因此适应动态 环境。 DGRS似乎提供了类似的好处。 DGRS通过 基本上与脊椎动物免疫系统不同。在DGR中，多元化源于 通用信息是针对一种特定基础，腺嘌呤和忠实于其他基础的。 这发生在遗传信息从RNA到cDNA的逆转录过程中，以及 腺嘌呤塑造蛋白质功能变异的模式。这种对腺丝的选择性不忠是中心 DGRS的标志性功能。选择性不忠在生物学上是独一无二的，以及如何发生未知。我们做了 通过在体外重新建立特定的不忠行为，对这个问题的显着突破 Bordetella uinaophage dgr。我们发现DGR逆转录酶BRT与 DGR附属可变性确定剂（AVD）蛋白是必需的，足以合成腺嘌呤 诱变的cDNA。我们的结果表明，BRT-AVD和DGR RNA合并形成功能和 结构化核糖核蛋白（RNP）颗粒。我们正处于揭示分子和原子的边缘 通过以下特定目的，选择性不忠的细节：（1）可视化BRT-AVD/RNA 通过冷冻电子显微镜在不同功能状态下的复合物； （2）确定核碱酶和 蛋白质确定负责选择性不忠行为； （3）确定位置的基础 - 特定不忠的特定调制。我们提出的研究将为 了解DGR。此外，由于选择性不忠的独特性，这些研究也将 可能会提供对控制其他逆转录酶（例如HIV RT）的忠诚度的机制的新见解。 一般而言，核苷酸聚合酶。