Mechanism of Vaccinia Virus Transcription Termination

痘苗病毒转录终止机制

• 批准号: 7018414
• 负责人: Edward G. Niles
• 金额: $ 34.49万
• 依托单位: STATE UNIVERSITY OF NEW YORK AT BUFFALO
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-07-15 至 2008-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7018414
• 关键词: DNA directed RNA polymerase; Poxviridae; adenosinetriphosphatase; conformation; enzyme linked immunosorbent assay; gene mutation; genetic transcription; laboratory rabbit; protein binding; protein structure function; regulatory gene; transcription factor; transcription termination; vaccinia virus; virus genetics

DESCRIPTION (provided by applicant): Early vaccinia virus gene transcription takes place in the virion core and is the only class that demonstrates signal dependent termination. Early gene transcription termination requires the signal UUUUUNU in the nascent RNA, VTF, the vaccinia termination factor that is also the viral mRNA capping enzyme, NPH I a single stranded DNA dependent ATPase to provide energy and the virion form of the multi-subunit RNA polymerase containing the additional H4L subunit. The goal of this research is to define the mechanism of early gene transcription termination. In this submission, we propose three specific aims. In Aim 1, we will focus on the role of NPH I in termination. We will determine whether NPH I binds to the nontemplate strand of the DNA to activate the ATPase activity. In addition, we will investigate the conformational change in NPH I induced upon ssDNA binding. Mutants in NPH I known to affect termination, in vitro, will be constructed in virus and their phenotypes investigated in vivo. Finally, the role of NPH I in transcription elongation will be evaluated. In Aim 2, our attention switches to VTF, the vaccinia termination factor. We will determine whether VTF is the factor that senses UUUUUNU in the nascent RNA. In addition, we will identify additional VTF interacting partners. Mutagenesis of VTF will permit identification of the termination specific functions. Finally, our novel observation of UUUUUNU specific activation of premature termination in trans will be fully explored. In Aim 3, we will address the role of the H4L subunit of the virion RNA polymerase in both early gene transcription initiation and termination. We propose to locate the NPH I binding site on H4L. We will map the region of H4L that is required for pre-initiation complex formation. We will search for H4L interacting proteins to both identify the factors required for PIC formation and additional proteins that may function in termination. Finally, we will construct mutations in virus that exhibit altered NPH I binding and evaluate their phenotypes in vivo. Through these studies we will define aspects of the mechanism of early gene transcription. These results will provide important insights into this essential step in gene expression and define new sites for the development of specific poxvirus antiviral agents.

描述(由申请人提供)：早期痘苗病毒基因转录发生在病毒核心，是唯一显示信号依赖终止的类别。早期的基因转录终止需要新生RNA中的信号UUUUUNU，VTF，痘苗终止因子，也是病毒mRNA封顶酶，NPH I，一种单链DNA依赖的ATPase来提供能量，以及含有额外H4L亚基的多亚单位RNA聚合酶的病毒形式。本研究的目的是明确早期基因转录终止的机制。在本意见书中，我们提出了三个具体目标。在目标1中，我们将重点介绍NPH I在终止妊娠中的作用。我们将确定NPH I是否与DNA的非模板链结合来激活ATPase活性。此外，我们还将研究单链DNA结合后NPH I的构象变化。已知在体外影响终止的NPH I突变体将在病毒中构建，并在体内研究其表型。最后，将评估NPH I在转录延伸中的作用。在目标2中，我们的注意力转向VTF，即牛痘终止因子。我们将确定VTF是否是在新生RNA中感觉UUUUUNU的因子。此外，我们将确定更多的VTF互动合作伙伴。VTF的诱变将允许识别终止的特定功能。最后，我们将对UUUUUNU对TRANS中提前终止的特异性激活的新观察进行充分的探索。在目标3中，我们将讨论病毒RNA聚合酶H4L亚基在早期基因转录启动和终止中的作用。我们建议将NPH I结合位点定位在H4L上。我们将绘制形成预引发复合体所需的H4L区域。我们将寻找H4L相互作用的蛋白质，以确定PIC形成所需的因素和可能在终止中发挥作用的其他蛋白质。最后，我们将构建显示NPH I结合改变的病毒突变，并在体内评估它们的表型。通过这些研究，我们将确定早期基因转录机制的各个方面。这些结果将为基因表达的这一关键步骤提供重要的见解，并为开发特定的痘病毒抗病毒药物定义新的位点。