Regulation of Gene Expression and the Cell Cycle

基因表达和细胞周期的调节

• 批准号: 6951639
• 负责人: DONALD COURT
• 金额: --
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6951639
• 关键词: Escherichia coli; bacterial RNA; bacterial genetics; bacterial proteins; bacteriophage lambda; cell cycle; cell growth regulation; gene expression; gene induction /repression; genetic recombination; genetic regulation; genetic transcription; genetic translation; host organism interaction; microorganism culture; molecular cloning; operon; plasmids; ribonuclease III; virus genetics; virus infection mechanism; virus protein

Developmental systems are controlled by modulating gene expression in response to internally programmed signals responding to external signals. Our laboratory is interested in studying the molecular interactions and the signaling that occur to regulate gene expression and the cell cycle. We exploit the genetic systems available in Escherichia coli, its plasmids, and its viruses (e.g., bacteriophage lambda) to help us understand (1) regulation at the levels of transcription initiation and elongation, translation initiation, and cell growth and cell cycle control signals and (2) Recombination and cloning using lambda Red functions. The N gene of lambda is the first gene expressed following viral infection. The function of the N protein is necessary for expression of most other lambda genes by its actions as a positive regulator. Positive activation of other genes occurs by N binding to specific RNA sites called NUT, modifying the RNA polymerase transcription complex. This modified polymerase complex reads through transcription terminators to distal lambda genes. Thus, the expression and action of N are central to the control of lambda development. We have recently determined that N is subject to novel posttranscriptional regulatory circuits. Expression of the N gene is autoregulated by N binding to the NUT RNA site 150 bases upstream of the N gene, from which the translation of N 100-fold over this long distance. The N-modified RNA polymerase complex is required for this translational repression. Thus, antitermination and translation repression by N are coupled. This may be caused by a specific folding of the RNA structure into a long duplex that brings the NUT RNA into close juxtaposition with the N ribosome binding site. RNaseIII, a ds RNA endonuclease, recognizes the stem structure and cleaves it, separating NUT from the N RNA. This cleavage prevents N translational repression but actually enhances antitermination, presumably by releasing the antitermination complex from its interaction with the NUT RNA. The degree of RNaseIII processing of the N-leader controls the amount of translation repression by N. Since RNaseIII expression itself is controlled by growth rate of the cells (see below), the cellular growth rate determines how much N protein will be made during a lambda infection. In rich media, high levels of RNaseIII exist in the cell thereby preventing any repression of N, while in poor media low levels of RNaseIII exist which is insufficient to prevent repression of N levels. The biology and development of lambda depends upon N levels which is modulated by RNaseIII and growth rate. Additionally, we have found that RNaseIII is expressed from an operon in which an essential low-molecular-weight GTP-binding protein, Era, is also encoded. From this operon, RNaseIII and Era expression is coordinately regulated and increases in relation to growth rate. This growth rate regulation of RNaseIII and Era occurs at the posttranscriptional level, but the mechanism remains unknown. The accumulation of adequate levels of Era is essential for cytokinesis to be completed and cell growth to continue. We speculate that a threshold level of Era must accumulate before Era-GTPase is activated by a cellular signal to cause cell division and to allow cell growth to continue. Era binds to precursor RNA and may use this binding as a measure of RNA synthesis and the signal to activate its GTPase. We believe RNaseIII and Era are key components that couple regulation of growth and the cell cycle. The crystal structure of both proteins has now been determined. A model for dsRNA cleavage by RNase III has been postulated from the structures. Because RNase III of eukaryotes is the active component in RNA-inhibition (RNAi), the structure and mechanism of cleavage by RNase III is of critical importance in understanding RNAi.

发育系统是通过调节基因表达来控制的，基因表达响应于内部编程信号，内部编程信号响应于外部信号。我们的实验室有兴趣研究分子相互作用和信号发生调节基因表达和细胞周期。我们利用大肠杆菌、其质粒和其病毒中可用的遗传系统（例如，噬菌体λ）来帮助我们理解（1）在转录起始和延伸、翻译起始以及细胞生长和细胞周期控制信号水平上的调节，以及（2）使用λ Red功能的扩增和克隆。 λ的N基因是病毒感染后表达的第一个基因。N蛋白的功能对于大多数其他λ基因的表达是必需的，其作为正调节剂的作用。其他基因的正激活通过N结合到称为NUT的特定RNA位点，修饰RNA聚合酶转录复合物而发生。这种修饰的聚合酶复合物通过转录终止子读取到远端λ基因。因此，N的表达和作用是控制lambda发育的核心。我们最近已经确定，N是受新的转录后调控电路。N基因的表达是通过N结合到N基因上游150个碱基的NUT RNA位点来自动调节的，从该位点N在该长距离上翻译100倍。N-修饰的RNA聚合酶复合物是这种翻译抑制所必需的。因此，N的抗终止和翻译阻遏是偶联的。这可能是由于RNA结构特异性折叠成长双链体，使NUT RNA与N核糖体结合位点紧密并列。RNA酶III是一种双链RNA内切酶，识别茎结构并将其切割，将NUT与N RNA分离。这种切割阻止了N翻译抑制，但实际上增强了抗终止，大概是通过释放抗终止复合物与NUT RNA的相互作用。RNA酶III对N前导序列的加工程度控制着N对翻译的抑制程度。由于RNaseIII表达本身受细胞生长速率控制（见下文），因此细胞生长速率决定了λ感染期间将产生多少N蛋白。在富培养基中，细胞中存在高水平的RNase III，从而防止N的任何抑制，而在贫培养基中存在低水平的RNase III，其不足以防止N水平的抑制。λ的生物学和发育取决于由RNaseIII和生长速率调节的N水平。 此外，我们还发现RNaseIII是由一个操纵子表达的，其中还编码一种必需的低分子量GTP结合蛋白Era。从这个操纵子，RNaseIII和Era的表达是协调调节和增加有关的增长率。RNaseIII和Era的这种生长速率调节发生在转录后水平，但其机制尚不清楚。足够水平的Era的积累对于胞质分裂的完成和细胞生长的继续是必不可少的。我们推测Era的阈值水平必须在Era-GTdR被细胞信号激活以引起细胞分裂并允许细胞生长继续之前积累。Era与前体RNA结合，并可利用这种结合作为RNA合成的量度和激活其GT3的信号。我们认为RNaseIII和Era是生长和细胞周期调控的关键组成部分。这两种蛋白质的晶体结构现已确定。从结构中推测了RNA酶III切割dsRNA的模型。由于真核生物的RNase III是RNA抑制（RNAi）的活性成分，因此RNase III的结构和切割机制对于理解RNAi至关重要。