NUCLEIC ACID STRUCTURE/FUNCTION IN GENE EXPRESSION

该提案的长期目标是在分子水平上了解 信使合成、加工和翻译的某些方面 大肠杆菌中的RNA。 重点是确定监管 在这些过程中进行控制。 平移耦合 将分析丝状噬菌体的基因V和VII之间的关系， 重点是理解V区严重下调的基础， VII顺反子间连接和识别偶联的决定因素 效率和与核糖体组分可能的相互作用。 的 将在体内研究翻译偶联的机制， 随后在体外，使用16 S rRNA突变体与改变的抗Shine- Dalgarno序列指导壮观霉素抗性核糖体进入 上游顺反子带有互补的Shine-Dalgarno序列。 的 所用的偶联起始位点将是在早期工作中研究的那些， 缓慢没有独立的活动和功能，只有当立即下游 一个翻译的区域。 内切核酸裂解加工事件， 产生大量的主要噬菌体f1 mRNA将被进一步研究， 重点关注作为RNase E可能实例的ams/rne依赖性切割 裂解位点 由于这些RNA的产生并不发生在 温度敏感的AMS/RNE突变体，加工对 现在可以探索噬菌体基因表达或生命周期。 作为 使用切割缺陷型EcoRI核酸内切酶突变体的研究结论 作为转录路障和延伸复合物结构的探针 和功能，大肠杆菌RNA聚合酶沿着DNA模板沿着移动将是 考察

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.