Structural studies of bacteriophage genome packaging and ejection

噬菌体基因组包装和排出的结构研究

• 批准号: 9241508
• 负责人: Reginald McNulty
• 金额: $ 6万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-04-01 至 2017-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9241508
• 关键词: 3-Dimensional; ATP Hydrolysis; Address; Adenoviruses; African American; Animals; Antibiotic Resistance; Appointment; Bacterial Infections; Bacteriophages; Binding; Capsid; Case Study; Cells; Cessation of life; Chickens; Complex; Contracts; Cytoplasm; Data; Disease; Disease Outbreaks; Double Stranded DNA Virus; Drug Design; Educational process of instructing; Electron Microscopy; Ethnic group; Funding; Genome; Goals; HIV; HIV-2; Health; Herpesviridae; Hispanics; Holoenzymes; Hospitalization; Human; Human Herpesvirus 2; Infection; Injection of therapeutic agent; Location; Lytic Virus; Membrane; Modeling; Molecular; Motor Activity; Needles; Patients; Podoviridae; Positioning Attribute; Poxviridae; Proteins; Research; Research Training; Resolution; Risk; Route; Salmonella; Simplexvirus; Site; Structure; Surface; Tail; Time; United States; Universities; Viral Proteins; Virulent; Virus; Virus Diseases; Woman; bacteriophage tailspike protein; base; career; density; ds-DNA; genital herpes; particle; pathogen; post-doctoral training; receptor; seal; stoichiometry; terminase; three dimensional structure

DESCRIPTION (provided by applicant): The Salmonella-infecting bacteriophage P22 packages its genome from concatemers of dsDNA. This packaging machinery consists of a large (L-) and small (S-) terminase complex. The small terminase functions to properly position the large terminase for packaging initiation via the pac site. The large terminase uses ATP hydrolysis to translocate a single copy of the genome into the bacteriophage procapsid. The motor activity of L-terminase results in dsDNA packaging at a rate of up to 2000 bp/sec. Although poorly understood, termination and cleavage are induced upon reaching a filled procapsid. Upon cleavage, the L-/S-terminase complex quickly disassociates from the capsid enabling tail and associated proteins to bind, sealing the genome inside the procapsid. Upon binding to surface receptors of Salmonella, dsDNA along with ejection proteins (gp7, gp16, and gp20) are injected to host cytoplasm. These ejection proteins are required for correct delivery of dsDNA to host cytoplasm. Aim 1 will [use single particle electron microscopy] elucidate the 3D structure and stoichiometry of L-/S- terminase holoenzyme; a state just prior to binding to procapsid. [Current proposed mechanisms for terminase mechanism is based on inconclusive, low-resolution, 34 angstrom data. Resolution better than 7A will resolve mechanism controversies in the field.] Aim 2 will characterize the 3D structure of P22 with its terminase complex bound; a state just prior to genome packaging. Aim 3 will capture the structure of genome and gp26 (tail needle) deficient P22; a state just after genome ejection. We have preliminary [single particle] data of a gp26 minus phage that shows extra density where the tail needle used to be. It is our hypothesis that an ejection (gp7, gp16, or gp20) contributes to this density. Together, the aims will enable new hypotheses to be developed which will elucidate the mechanism of genome packaging and injection into the host

描述（由申请人提供）：感染沙门氏菌的噬菌体P22从dsDNA的多联体包装其基因组。这种包装机器由大（L-）和小（S-）末端酶复合物组成。小末端酶的功能是正确定位大末端酶，以通过pac位点启动包装。大末端酶利用ATP水解将基因组的单个拷贝移位到噬菌体原衣壳中。L-末端酶的马达活性导致dsDNA以高达2000 bp/sec的速率包装。虽然知之甚少，但终止和切割是在到达充满的原衣壳时诱导的。切割后，L-/S-末端酶复合物迅速与衣壳分离，使尾部和相关蛋白结合，将基因组密封在原衣壳内。一旦与沙门氏菌的表面受体结合，dsDNA沿着与排出蛋白（gp 7、gp 16和gp 20）一起被注射到宿主细胞质中。这些喷射蛋白是将dsDNA正确递送到宿主细胞质所需的。目标1将[使用单粒子电子显微镜]阐明L-/S-末端酶全酶的3D结构和化学计量;一种刚好在结合到原衣壳之前的状态。[目前提出的终止酶机制是基于不确定的、低分辨率的34埃数据。高于7A的分辨率将解决该领域的机制争议。目的2将表征P22的3D结构及其末端酶复合物结合;基因组包装之前的状态。目的3将捕获基因组和gp 26（尾针）缺陷P22的结构;基因组排出后的状态。我们有一个gp 26负噬菌体的初步[单粒子]数据，显示了尾针曾经存在的额外密度。我们的假设是，一个喷射（gp 7，gp 16，或gp 20）有助于这个密度。总之，这些目标将使新的假设得以发展，这将阐明基因组包装和注入宿主的机制