Mechanism of Poly(a) Tail Formation by Vaccinia Virus

痘苗病毒形成 Poly(a) 尾的机制

• 批准号: 7267766
• 负责人: Paul D Gershon
• 金额: $ 26.03万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-08-01 至 2008-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7267766
• 关键词: Address; Affinity; Atomic Force Microscopy; Binding Sites; Biological; Biological Assay; Boxing; Catalysis; Collaborations; Complex; Coupled; Enzymes; Flexor; Fluorescence; Fluorescence Resonance Energy Transfer; Funding; Human Resources; Individual; Length; Ligands; Mass Spectrum Analysis; Messenger RNA; Modification; Molecular; Movement; Nucleic Acids; Nucleotides; Phosphorylation; Poly(A) Tail; Polyadenylation; Polymerase; Polynucleotide Adenylyltransferase; Positioning Attribute; Process; Property; Proteins; RNA; RNA Splicing; Signal Transduction; Structure; Structure-Activity Relationship; Summary Reports; Tail; Techniques; Vaccinia; Vaccinia virus; crosslink; insight; molecular dynamics; mutant; research study; single molecule; stop flow technique; stopped-flow fluorescence; structural biology; three dimensional structure; tool

DESCRIPTION (provided by applicant): Although the poly (A) tail is a nearly ubiquitous feature of mRNA, the structure and dynamics of the enzyme that adds it are largely a black box. The vaccinia virus poly (A) polymerase (PAP) appears to be unique among the non-templated nucleic acid polymerases in possessing (a) an innate ability to translocate, (b) a processivity factor that can assist the polymerase in elongation. Moreover, the vaccinia enzyme is unusual in adding a tail in the absence of coupled processes such as RNA cleavage signal recognition, RNA endonucleolytic scission, mRNA splicing and protein phosphorylation. The vaccinia virus PAP therefore provides a powerful tool with which to study important aspects of polymerase molecular dynamics within a single-chain enzyme. Although the isolated catalytic (VP55) subunit of the vaccinia PAP heterodimer can processively elongate a primer, polyadenylation halts after tails approximately 25 - 30 nt in length have been added. The VP39 subunit is a processivity factor for tail elongation by VP55. VP39's structure and properties were studied during the initial funding period. The second funding period continued with a characterization of the translocational properties of the VP55 subunit, and the topography of both the heterodimer and the heterodimer-RNA ternary complex. Very recently, a long-standing obstacle to the study of VP55, namely its high-level expression was overcome, opening the door to structural and functional approaches requiring larger amounts of VP55 protein. Aim I of this competing continuation addresses the structural biology and structure-function relationships within the polymerase-processivity factor (VP55-VP39) heterodimer. Aims 2 and 3 address polymerase molecular dynamics. It is proposed that techniques in use in the P.l.'s lab (molecular biological approaches, photo crosslinking, mass spectrometry) will be augmented by inter-lab collaboration for the application of stopped-flow fluorescence and atomic force microscopy. It is expected that the proposed approaches will provide an integrated view of the structure and dynamics of a translocating, non-templated polymerase during formation of the poly (A) tail.

描述（由申请人提供）：虽然poly（A）尾几乎是mRNA的普遍特征，但添加它的酶的结构和动力学在很大程度上是一个黑盒。牛痘病毒多聚腺苷酸聚合酶（PAP）在非模板核酸聚合酶中似乎是独特的，具有（a）先天易位能力，（B）可帮助聚合酶延伸的持续合成因子。此外，牛痘酶在不存在偶联过程如RNA切割信号识别、RNA内切核酸裂解、mRNA剪接和蛋白质磷酸化的情况下添加尾部是不寻常的。因此，牛痘病毒PAP提供了一个强有力的工具，研究聚合酶分子动力学的重要方面的单链酶。尽管牛痘PAP异二聚体的分离的催化（VP 55）亚基可促发性延长引物，但在添加了约25 - 30 nt长的尾部后，聚腺苷酸化停止。VP 39亚基是VP 55尾延伸的持续合成因子。VP 39的结构和性质在最初的资助期间进行了研究。第二个资助期继续进行VP 55亚基的易位特性的表征，以及异二聚体和异二聚体-RNA三元复合物的拓扑结构。最近，VP 55研究的一个长期障碍，即其高水平表达被克服，为需要更大量VP 55蛋白的结构和功能方法打开了大门。目的我的竞争继续地址的结构生物学和结构功能的关系内的聚合酶-合成因子（VP 55-VP 39）异二聚体。目的2和3解决聚合酶分子动力学。建议在P.I.的实验室（分子生物学方法，光交联，质谱法）将通过实验室间的合作来加强停流荧光和原子力显微镜的应用。预计所提出的方法将提供在poly（A）尾形成期间易位、非模板聚合酶的结构和动力学的综合视图。

项目成果

Domain-level rocking motion within a polymerase that translocates on single-stranded nucleic acid.

在单链核酸上易位的聚合酶内的域水平摇摆运动。

• DOI: 10.1107/s0907444913000346
• 发表时间: 2013
• 期刊: Acta crystallographica. Section D, Biological crystallography
• 作者: Li,Huiyung;Li,Changzheng;Zhou,Sufeng;Poulos,ThomasL;Gershon,PaulDavid
• 通讯作者: Gershon,PaulDavid

Methyltransferase-specific domains within VP-39, a bifunctional protein that participates in the modification of both mRNA ends.

VP-39 内的甲基转移酶特异性结构域是一种双功能蛋白，参与 mRNA 两端的修饰。

• 发表时间: 1996
• 期刊: RNA (New York, N.Y.)
• 作者: Shi,X;Yao,P;Jose,T;Gershon,P
• 通讯作者: Gershon,P

Polymerase translocation with respect to single-stranded nucleic acid: looping or wrapping of primer around a poly(A) polymerase.

相对于单链核酸的聚合酶易位：引物在聚腺苷酸聚合酶周围形成环或包裹。

• DOI: 10.1016/j.str.2009.03.012
• 发表时间: 2009
• 期刊: Structure (London, England : 1993)
• 作者: Li,ChangZheng;Li,Huiying;Zhou,Sufeng;Sun,Eric;Yoshizawa,Janice;Poulos,ThomasL;Gershon,PaulD
• 通讯作者: Gershon,PaulD

Evidence that the RNA methylation and poly(A) polymerase stimulatory activities of vaccinia virus protein VP39 do not impinge upon one another.

有证据表明痘苗病毒蛋白 VP39 的 RNA 甲基化和聚腺苷酸聚合酶刺激活性不会相互影响。

• DOI: 10.1006/viro.1998.9209
• 发表时间: 1998
• 期刊: Virology.
• 作者: Gershon,PD;Shi,X;Hodel,AE
• 通讯作者: Hodel,AE

A polyadenylylation-specific RNA-contact site on the surface of the bifunctional vaccinia virus RNA modifying protein VP39 that is distinct from the mRNA 5' end-binding "cleft".

双功能痘苗病毒 RNA 修饰蛋白 VP39 表面上的聚腺苷酸化特异性 RNA 接触位点与 mRNA 5 末端结合“裂口”不同。

• DOI: 10.1006/jmbi.1998.2417
• 发表时间: 1999
• 期刊: Journal of molecular biology.
• 作者: Deng,L;Johnson,L;Neveu,JM;Hardin,S;Wang,SM;Lane,WS;Gershon,PD
• 通讯作者: Gershon,PD