NASCENT PEPTIDE CONTROL OF THE RIBOSOME

核糖体的新生肽控制

• 批准号: 2628367
• 负责人: PAUL S LOVETT
• 金额: $ 32.09万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE COUNTY
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-05-01 至 2002-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2628367
• 关键词: Bacillus subtilis; DNA footprinting; Escherichia coli; chloramphenicol; drug resistance; gene induction /repression; gene mutation; genetic translation; messenger RNA; peptide chemical synthesis; peptides; ribosomal RNA; ribosomes

Two-gene operons have been identified in bacteria and eukaryotes in which the nascent peptide product of the upstream gene interferes with a function(s) of its translating ribosome. This can activate or abolish translation of the downstream coding sequence depending on the gene system. In translation attenuation regulation of inducible chloramphenicol resistance genes in bacteria, ribosome stalling at a specific codon in the leader coding sequence (the upstream gene) destabilizes an adjacent region of mRNA secondary structure that sequesters the ribosome binding site for the downstream coding sequence, which specifies the antibiotic resistance protein. thus, ribosome stalling in the upstream coding sequence activates translation of the downstream coding sequence. We propose the pivotal first two steps of a three step model for translation attenuation depend on cis-interaction of the nascent leader peptide with its translating ribosome. Mutations in 23S rRNA that prevent Step 1 (the binding of peptide to rRNA) will be tested for induction in vivo and for their effect on peptide inhibition of peptidyltransferase. Ribosome pausing in the leader due to Step 1 is converted to ribosome stalling (Step 2) in the presence of inducer. We suggest that the nascent peptide alters the response of the target ribosome to chloramphenicol. This will be tested by identifying the ribosomal components altered during the combined addition of peptide and inducer, and by in vivo analysis of translational fusions of the leader to lacZ in bacteria and yeast. Step 3 is proposed to result from an interaction of the rRNA with mRNA nucleotides in the leader proximal region of secondary structure. This will be examined by experiments that swap expressed 23S rRNA's and by modifications in the sequence of the secondary structures that regulate translation attenuation. The amino acid sequence of the leader peptides dictates 23S rRNA binding activity. We have identified a simple method that should allow determination of the critical amino acids for binding. Further, we will refine the identification of the rRNA site for peptide binding by high resolution footprinting.

已在细菌和真核生物中鉴定出双基因操纵子 上游基因的新生肽产物会干扰哪些 其翻译核糖体的功能。 这可以激活或取消 下游编码序列的翻译取决于基因 系统。 诱导型翻译衰减调节 细菌中的氯霉素抗性基因，核糖体停滞在 前导编码序列（上游基因）中的特定密码子 使 mRNA 二级结构的相邻区域不稳定 隔离下游编码序列的核糖体结合位点， 其中指定了抗生素抗性蛋白。 因此，核糖体 上游编码序列的停滞激活了 下游编码序列。 我们提出关键的前两个步骤 翻译衰减的三步模型取决于顺式相互作用 新生前导肽及其翻译核糖体的结构。 突变 在 23S rRNA 中，阻止步骤 1（肽与 rRNA 的结合）将 测试体内诱导及其对肽的影响 肽基转移酶的抑制。 核糖体在前导区暂停 在存在以下物质的情况下，步骤 1 转化为核糖体停滞（步骤 2） 诱导剂。 我们认为新生肽改变了 将核糖体靶向氯霉素。 这将通过识别来测试 联合添加肽期间核糖体成分发生改变 和诱导剂，并通过翻译融合的体内分析 细菌和酵母中 lacZ 的领导者。 建议步骤 3 的结果是 rRNA 与近端前导序列中 mRNA 核苷酸的相互作用 二级结构区域。 这将通过实验来检验 交换表达 23S rRNA 并通过序列修改 调节翻译衰减的二级结构。 这 前导肽的氨基酸序列决定 23S rRNA 结合 活动。 我们已经确定了一种简单的方法，应该允许 确定结合的关键氨基酸。 此外，我们将 通过高通量优化对肽结合的 rRNA 位点的识别 分辨率足迹。