Regulation of Gene Expression and the Cell Cycle

发育系统是通过调节基因表达来控制的，以响应内部编程信号对外部信号的响应。我们实验室感兴趣的是研究调节基因表达和细胞周期的分子相互作用和信号转导。我们利用大肠杆菌、其质粒和其病毒(例如，噬菌体lambda)中可用的遗传系统来帮助我们理解(1)在转录起始和延伸、翻译起始、细胞生长和细胞周期控制信号水平上的调控，以及(2)使用lambda Red功能的重组和克隆。 Lambda的N基因是病毒感染后表达的第一个基因。N蛋白的功能是大多数其他lambda基因表达所必需的，因为它是一个正调控因子。其他基因的正激活是通过N与称为NUT的特定RNA位点结合，修改RNA聚合酶转录复合体来实现的。这个修饰过的聚合酶复合体通过转录终止子读取远端的lambda基因。因此，N的表达和作用是控制lambda发育的中心。我们最近确定N受制于新的转录后调控回路。N基因的表达是通过与N基因上游的NUT RNA位点150碱基的N结合来自动调节的，从那里N的翻译在这一长距离上增加了100倍。这种翻译抑制需要N-修饰的RNA聚合酶复合体。因此，反终止和N的翻译抑制是相结合的。这可能是由于RNA结构被折叠成一个长的双链，使NUT RNA与N核糖体结合位点紧密并列所致。RNaseIII是一种双链RNA内切酶，识别茎结构并将其切割，将NUT从N RNA中分离出来。这种切割阻止了N翻译抑制，但实际上增强了抗终止，可能是通过释放抗终止复合体与NUT RNA相互作用。N-先导的RNaseIII处理程度控制N的翻译抑制量。由于RNaseIII的表达本身由细胞的生长速度控制(见下文)，细胞的生长速度决定了在lambda感染期间将产生多少N蛋白。在富介质中，高水平的RNaseIII存在于细胞中，从而阻止了对N的任何抑制，而在差的介质中，存在低水平的RNaseIII，这不足以防止对N水平的抑制。Lambda的生物学和发育依赖于受RNaseIII和生长速率调控的N水平。 此外，我们还发现RNaseIII是从操纵子表达的，在操纵子中，一个必要的低分子质量的GTP结合蛋白Era也被编码。从这个操纵子开始，RNaseIII和Era的表达受到协同调控，并且随着生长速度的增加而增加。RNaseIII和Era的这种生长速度调节发生在转录后水平，但机制尚不清楚。足够水平的ERA的积累对于细胞质分裂的完成和细胞继续生长是必不可少的。我们推测，在ERA-GTP酶被细胞信号激活以引起细胞分裂并允许细胞继续生长之前，必须积累一个阈值水平的ERA。Era与前体RNA结合，并可能利用这种结合作为RNA合成的量度和激活其GTP酶的信号。我们认为RNaseIII和ERA是将生长调控和细胞周期结合在一起的关键成分。现在已经确定了这两种蛋白质的晶体结构。从这些结构中推测了RNaseIII切割dsRNA的模型。由于真核生物的RNase III是RNA抑制(RNAi)中的活性成分，RNase III的结构和切割机制对理解RNAi具有至关重要的作用。

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.