NUCLEIC ACID STRUCTURE/FUNCTION IN GENE EXPRESSION

我们的长期目标是了解关键的监管控制 在信使的处理和翻译过程中进行操作 大肠埃希氏菌中的RNA。目前的工作重点是 丝状菌主要转录单位中使用的机制 噬菌体实现精心平衡的基因表达模式 这使得其宿主中的持续感染得以维持。 首先，在F1基因V和VII提供的基因对中，基本 我们将测试平移耦合假说，重点是 在体内演示这一机制。方法是使用 具有反Shine-Dalgarno序列改变的16S rRNA突变体 选择性地引导耐壮观霉素核糖体进入 上游基因，已经提供了Shine-Dalgarno 与突变体16S rRNA互补的序列。基因VII是 是研究的理想之选，因为我们已经证明了它本身就是 单独出现时处于非活动状态。第二，既然我们已经获得了 有证据表明，位于 转录单位的近端是由目前为止 未知的RNe依赖的切割和翻译控制， 他们的监管基础将得到进一步界定。这双 基因更普遍地代表了一种在很大程度上未被探索的 原核生物中的一种策略，其功能与 真核生物中两种或两种以上的选择性剪接 来自同一基因的不同大小的蛋白质。第三，一个非常大的 在相关噬菌体中发现强翻译起始点 艾克将被认为是一个有希望的候选人 主要通过翻译增强剂发挥作用。从长远来看 这一目的的基本原理是将尚未确定的mrna-rrna定义为 调节引发复合体组装的相互作用 有助于目前完善16S rRNA结构的努力。

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.