CONTROL OF GENE EXPRESSION AND THE CELL CYCLE

发育系统是通过调节基因表达来响应内部编程信号对外部信号的响应来控制的。我们的实验室主要研究分子间的相互作用以及调控基因表达和细胞周期的信号传导。我们利用大肠杆菌、其质粒和病毒（如噬菌体l）中可用的遗传系统来帮助我们理解(1)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；(2)利用l Red功能进行重组和克隆。l的N基因是病毒感染后首先表达的基因。N蛋白的功能是大多数其他l基因表达所必需的，因为它是一个正调节因子。其他基因的正激活发生在N结合到特定的RNA位点上，称为NUT，修饰RNA聚合酶转录复合物。这种修饰的聚合酶复合体通过转录终止子读取远端基因。因此，N的表达和作用是控制l发育的核心。我们最近确定N受新的转录后调控回路的影响。N基因的表达是由N与上游150个碱基的NUT RNA位点结合而自动调节的，在这个距离上，N的翻译量是原来的100倍。这种翻译抑制需要n修饰的RNA聚合酶复合物。因此，反终止和N的平移抑制是耦合的。这可能是由于RNA结构的特定折叠成长双工，使NUT RNA与N核糖体结合位点紧密并置。RNaseIII是一种ds RNA内切酶，它识别茎结构并将其切割，将NUT从N RNA中分离出来。这种切割防止了N的翻译抑制，但实际上增强了抗终止，可能是通过释放与NUT RNA相互作用的抗终止复合体。此外，我们发现RNaseIII是由一个操纵子表达的，在这个操纵子中，一个重要的低分子量gtp结合蛋白Era也被编码。通过这个操纵子，RNaseIII和Era的表达被协调调节，并随着生长速率的增加而增加。RNaseIII和Era的生长速率调控发生在转录后水平，但其机制尚不清楚。足够水平的Era的积累是完成细胞质分裂和细胞继续生长所必需的。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继续生长之前，Era必须积累一个阈值水平。Era与前体RNA结合，并可能利用这种结合作为RNA合成的测量和激活其GTPase的信号。我们认为RNaseIII和Era是调控生长和细胞周期的关键成分。

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 l 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 l Red functions. The N gene of l is the first gene expressed following viral infection. The function of the N protein is necessary for expression of most other l 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 l genes. Thus, the expression and action of N are central to the control of l 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.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.