CELLULAR TRANS-ACTING FACTORS MEDIATING RSV ENCHANCER

介导 RSV 增强剂的细胞反式作用因子

• 批准号: 2180058
• 负责人: LINDA SEALY
• 金额: $ 26.27万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-04-01 至 1997-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2180058
• 关键词: DNA binding protein; HeLa cells; RNA binding protein; Rous sarcoma virus; biophysics; chemical binding; conformation; crosslink; fluorescence spectrometry; gel mobility shift assay; genetic enhancer element; genetic promoter element; genetic transcription; host organism interaction; immunoaffinity chromatography; immunoprecipitation; molecular cloning; nuclear runoff assay; nucleic acid repetitive sequence; nucleic acid sequence; posttranslational modifications; protein purification; protein reconstitution; protein sequence; protein structure function; recombinant proteins; site directed mutagenesis; transcription factor; transfection; virus genetics

The primary aim of our research is to define the molecular mechanisms by which the Rous sarcoma virus (RSV) long terminal repeat (LTR) enhancer activates transcription in a wide variety of eukaryotic cells. We have chosen to study the RSV LTR because this retrovirus appears to have been extremely adept at coopting cellular transcriptional machinery to promote its own expression. The enhancer and promotor elements located in the U3 region of the RSV LTR are among the strongest transactivating sequences identified in eukaryotic cells. Their potency and widespread function in many different cell types suggests that the transcription factors mediating LTR enhancer/promotor function will be essential proteins that normally play critical roles in regulating cellular gene expression. Consequently, understanding LTR enhancer function may provide valuable insights into the complex transcriptional regulatory network governing cell growth and/or differentiation that when altered, leads to neoplastic transformation. The molecular picture of the RSV LTR enhancer that has emerged from our studies over the past four years certainly supports this prediction. The major players mediating LTR enhancer function are a ubiquitously expressed, multi-component CCAAT-binding complex (CBC), the serum response factor (SRF), one or more members of the C/EBP family of transcription factors, and Myc, most likely with its partner Max. We propose that the LTR enhancer can be viewed as a bipartite structure. Transactivation is mediated by the distal one/third of the enhancer which binds C/EBP-related factors or Myc/Max, depending upon the relative balance of these transcription factors in the host cell. The CCAAT- binding complex and SRF, binding to the proximal two/thirds of the enhancer, create a specific protein-DNA architecture which conducive to high level enhancer function because it brings the more distally bound transactivators and basal transcription initiation machinery into close proximity. Research designed to test the validity of this view will be focused on (1) defining the protein composition of the multi-component CBC through reconstitution with recombinant proteins, (2) characterizing the RNA and DNA binding properties of a putative member of this complex, EFI/A, as well as pursuing state-of-the-art biophysical techniques to analyze the protein-DNA structure created when two CBCs and SRF bind to the proximal segment of the LTR enhancer in vitro, (3) positive identification of the C/EBP-related EFII factors via microprotein sequencing, including confirmation of preliminary data suggesting EFIIa is LIP and (4) further analysis of Myc and Max binding to the EFII cis element in vitro and potentially, in vivo. Information gained in these areas will provide a better understanding of how these various factors work together to achieve very high levels of transcriptional activation mediated by the LTR enhancer. At the same time, valuable information on the function of several critical regulators of cellular gene expression can be expected.

我们研究的主要目的是通过 Rous肉瘤病毒（RSV）长时间重复（LTR）增强子 激活多种真核细胞中的转录。 我们有 选择研究RSV LTR，因为该逆转录病毒似乎已经 非常擅长使用细胞转录机械来促进 它自己的表达。 位于 RSV LTR的U3区域是最强的反式激活之一 在真核细胞中鉴定出的序列。 他们的效力和广泛 在许多不同的细胞类型中的功能表明转录 介导LTR增强子/启动子功能的因素至关重要 通常在调节细胞基因中起关键作用的蛋白质 表达。 因此，了解LTR增强子功能可能 提供有关复杂转录调节的宝贵见解 网络管理细胞生长和/或分化时，改变时， 导致肿瘤转化。 从我们的RSV LTR增强器的分子图片 在过去的四年中，研究无疑支持这一预测。 这 介导LTR增强器功能的主要参与者普遍存在 表达，多组分CCAAT结合复合物（CBC），血清 响应因素（SRF），C/EBP家族的一个或多个成员 转录因子和MYC，最有可能具有其合作伙伴最大。 我们 提出可以将LTR增强剂视为两部分结构。 反式激活是由增强子的远端/三分之一介导的 绑定与C/EBP相关因素或MYC/MAX，具体取决于相对 这些转录因子在宿主细胞中的平衡。 CCAAT- 结合复合物和SRF，与近端两/三分之二的结合 增强剂，创建一个特定的蛋白质-DNA结构，有利于 高级增强器功能，因为它带来了更远端的界限 反式激活器和基础转录启动机械近距离 接近。 旨在测试这种观点有效性的研究将集中在 （1）通过定义多组分CBC的蛋白质组成 用重组蛋白重组，（2）表征RNA和 EFI/A的假定成员的DNA结合特性AS 以及追求最先进的生物物理技术来分析 当两个CBC和SRF与近端结合时，蛋白-DNA结构产生 LTR增强子在体外的段，（3） C/EBP相关的EFII因子通过微蛋白测序，包括 确认初步数据表明EFIIA是唇部，（4） 分析MYC和最大结合在体外与EFII CIS元件的结合， 可能，体内。 在这些领域获得的信息将提供 更好地了解这些各种因素如何共同起作用 达到由很高的转录激活介导的转录激活 LTR增强剂。 同时，有关功能的宝贵信息 可以预期几个关键的细胞基因表达调节剂。