NUCLEIC ACID STRUCTURE-FUNCTION IN GENE EXPRESSION

该提案的长期目标是在分子水平上理解 信使的合成、加工和翻译的某些方面 大肠埃希氏菌中的RNA。重点放在确定监管机构 在这些过程中运行的控件。翻译的耦合 将分析丝状噬菌体的基因V和VII之间的关系， 强调了解V-A严重下调监管的基础 Vii顺反子间连接和识别耦合的决定因素 效率和可能与核糖体成分的相互作用。这个 平移耦合的机制将在体内和 随后，在体外，使用一个16S rRNA突变体和一个改变的反光泽- Dalgarno序列将壮观霉素抗性核糖体定向进入 一个上游顺反子，带有免费的肖恩-达尔加诺序列。这个 所使用的偶联引发点将是在早期工作中研究的那些 减慢没有独立的活动，只有在立即下行时才起作用 翻译后的区域的。内核溶解处理事件 产生一些主要的噬菌体F1 mRNAs将被进一步研究， 关注依赖AMS/RNe的切割作为RNaseE的可能例子 裂解位点。因为这些RNA的产生不会发生在 温度敏感型AMS/RNe突变体的加工调控作用 现在可以探索噬菌体基因的表达或生命周期。作为一名 结论利用裂解缺陷型EcoRI内切酶突变体进行研究 作为转录障碍和延伸复合体结构的探针 和功能，大肠杆菌RNA聚合酶将沿着DNA模板移动 检查过了。

The long term objective of the proposal is to understand at the molecular certain aspects of the synthesis, processing and translation of messenger RNAs in Escherichia coli. Emphasis is placed on identifying regulatory controls that operate during these processes. Coupling of translation between genes V and VII of the filamentous phage will be analyzed, with emphasis on understanding the basis for the severe downregulation at the V- VII intercistronic junction and identifying determinants of coupling efficiency and possible interactions with ribosomal components. The mechanism of translational coupling will be studied in vivo and subsequently in vitro, using a 16S rRNA mutant with an altered anti-Shine- Dalgarno sequence to direct entry of a spectinomycin-resistant ribosome to an upstream cistron bearing the complimentary Shine-Dalgarno sequence. The coupled initiation sites used will be those studied in earlier work which slow no independent activity and function only when immediately downstream of a translated region. The endonucleolytic processing events that generate a number of the major phage f1 mRNAs will be studied further, focusing on the ams/rne-dependent cleavages as likely examples of RNase E cleavage sites. Since production of these RNAs does not occur in temperature-sensitive ams/rne mutants, the regulatory role of processing on phage gene expression or the life cycle can now be explored. As a conclusion to studies using cleavage-defective EcoRI endonuclease mutants as transcriptional roadblocks and probes of elongation complex structure and function, E.coli RNA polymerase movement along the DNA template will be examined.