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.

发展系统是通过对响应外部信号的内部编程信号调节基因表达来控制的。我们的实验室有兴趣研究分子相互作用以及调节基因表达和细胞周期的信号传导。我们利用大肠杆菌，其质粒及其病毒（例如噬菌体lambda）中可用的遗传系统，以帮助我们理解（1）在转录启动和伸长启动，翻译起始，翻译启动以及细胞生长和细胞周期对照信号以及使用Lambda红色功能的调节。 lambda的n基因是病毒感染后第一个表达的基因。 N蛋白的功能对于大多数其他lambda基因作为阳性调节剂的作用是必要的。其他基因的阳性激活是通过N结合与称为螺母的特定RNA位点发生的，从而改变了RNA聚合酶转录复合物。这种修饰的聚合酶复合物通过转录终止剂读取远端lambda基因。因此，N的表达和作用对于控制Lambda发育的控制至关重要。我们最近确定n受到新的转录后调节电路的约束。 n基因的表达是通过n结合N基因上游的Nut RNA位点150碱基的自动调节的，该基因在该基因的上游底部的表达是在该基因上的上游，在该基因的长距离内将N 100倍转移。这种翻译抑制需要N修饰的RNA聚合酶复合物。因此，n耦合抗性和翻译抑制。这可能是由于RNA结构的特异性折叠引起的，将螺母RNA与N核糖体结合位点近来并置。 DS RNA核酸内切酶RNAseIII识别茎结构并裂解，将螺母与N RNA分开。这种裂解阻止了N转化抑制，但实际上可以增强抗抑制作用，大概是通过释放抗抑制剂复合物与螺母RNA的相互作用。 N-Leader的RNaseIII处理程度控制N。由于RNaseIII表达本身受细胞的生长速率控制（见下文），因此细胞生长速率决定了Lambda感染期间将产生多少N蛋白。在丰富的培养基中，细胞中存在高水平的RNAseIII，从而防止了n抑制N，而在较差的培养基中，RNAseIII的含量较低，这不足以防止抑制N水平。 Lambda的生物学和发展取决于n个水平，该水平受RNaseIII和生长速率调节。 此外，我们发现rnaseiii是根据操纵子表达的，其中必需的低分子量GTP结合蛋白ERA也被编码。从该操纵子中，RNAseIII和ERA表达受到协调的调节，并且与增长率有关。 RNaseIII和ERA的这种增长率调节发生在转录后水平，但该机制仍然未知。适当水平的ERA的积累对于完成的细胞因子和细胞生长继续至关重要。我们推测，在ERA-GTPase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，必须积累ERA的阈值水平。 ERA与前体RNA结合，并可以将此结合用作RNA合成的度量和激活其GTPase的信号。我们认为，rnaseiii和ERA是调节生长和细胞周期的关键组成部分。现在已经确定了两种蛋白质的晶体结构。 RNase III的DSRNA裂解模型已从结构中假设。由于真核生物的RNase III是RNA抑制（RNAI）的活性成分，因此RNase III裂解的结构和机理对于理解RNAi至关重要。