NUCLEIC ACID STRUCTURE/FUNCTION IN GENE EXPRESSION

我们的长期目标是了解关键的监管控制 在处理和翻译信使的过程中运作， 大肠杆菌中的RNA。 目前的工作重点是 在丝状体的主要转录单位中使用的机制 噬菌体，以实现基因表达的仔细平衡模式 使其在宿主体内持续感染。 首先，在由f1基因V和VII提供的基因对中， 翻译耦合的假设将被测试，重点是 在体内演示其机制。 方法是使用 一种16 S rRNA突变体，其抗Shine-Dalgarno序列发生改变 选择性地引导壮观霉素抗性核糖体进入， 上游基因，已提供了一个Shine-Dalgarno 与突变体16 S rRNA互补的序列。 基因七是 因为我们已经证明了它本质上 单独存在时不活跃。 第二，由于我们已经获得 有证据表明，这对框内重叠的基因在 转录单位的近端受到调控， 未知的rne依赖性切割和翻译控制， 将进一步明确其监管依据。 这双 基因更普遍地代表了一个很大程度上未被探索的 原核生物中的策略，其实现与 真核生物中的选择性剪接，产生两个或多个 来自同一基因的不同大小的蛋白质。 第三，非常 在相关噬菌体中发现的强翻译起始位点 IKe将被探索为一个有希望的候选人， 主要通过翻译增强子发挥作用。 的长期 这一目标的基本原理是定义尚未鉴定的mRNA-rRNA 介导起始复合物组装的相互作用， 为目前完善16 S rRNA结构的努力做出了贡献。

Our longterm objective is to understand key regulatory controls that operate during the processing and translation of messenger RNAs in Escherichia coli. Current work focuses on the mechanisms used in the major transcription unit of the filamentous phage to achieve the carefully balanced pattern of gene expression that permits a persistent infection in its hosts to be maintained. First, in the gene pair provided by f1 genes V and VII, the basic hypothesis for translational coupling will be tested, with emphasis on demonstrating the mechanism in vivo. The approach is to use a 16S rRNA mutant with an altered anti-Shine-Dalgarno sequence to direct entry of a spectinomycin-resistant ribosome selectively to the upstream gene, which has been provided a Shine-Dalgarno sequence complementary to the mutant 16S rRNA. Gene VII is ideal for the studies since we have shown it to be inherently inactive when present alone. Second, since we have obtained evidence that the pair of in-frame overlapping genes at the proximal end of the transcription unit is regulated both by as yet unknown rne-dependent cleavages and by translational control, the basis for their regulation will be defined further. This pair of genes is more generally representative of a largely unexplored strategy in prokaryotes which achieves the same function as alternative splicing in eukaryotes, production of two or more proteins of different size from the same gene. Third, a very strong translation initiation site discovered in the related phage IKe will be explored as a promising candidate for one that functions primarily through translational enhancers. The longterm rationale for this aim is to define as yet unidentified mRNA-rRNA interactions that mediate initiation complex assembly and thereby contribute to current efforts to refine the structure of 16S rRNA.