POWRE: Optimization and Characterization of Chloroplast Translation In Vitro

POWRE：叶绿体体外翻译的优化和表征

• 批准号: 9720444
• 负责人: Margaret Hollingsworth
• 金额: $ 7.49万
• 依托单位: SUNY at Buffalo
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-02-15 至 2001-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9720444&HistoricalAwards=false
• 关键词: POWRE; Optimization; Characterization; Chloroplast; Translation

9720444 Hollingsworth Regulation at the translational level is a key contol point in the expression of many choroplast genes. Such regulation is typically mediated through RNA-protein interactions at the 5' untranslated region of an mRNA. This laboratory has previously shown that specific RNA-protein interactions occur at the 5' untranslated regions of chloroplast-encoded ATP synthase genes. Proposals to analyze the effects of these interactions on translation in vitro have been stymied by reviewers' concerns about the technical challenges present by chloroplast translation extracts. The goal of this research is to answer those concerns by analyzing and optimizing a recently reported protocol for preparing chloroplast translation extracts. The project has two primary objectives: The first objective is to optimize the translation assay to expand the repertoire of transcripts that can be efficiently translated. Two transcripts with several orders of magnitude difference in translation efficiencies in vitro will be used as substrates for these experiments. Analysis of the effects of cis-acting sequences on translation will be initiated using chimerics of the two original substrates. The second objective of this research is to expand the applicability of the extract preparation of protocol. Experiments will be performed to expand the protocol from chloroplasts of tobacco to chloroplasts from other research-intensive species, including spinach and Arabidopsis. This project will be supported through the POWRE program. It will determine the feasibility of a new line of inquiry that should expand the applicability of the in vitro chloroplast translation to inquiries examining translational regulation of chloroplast-encoded ATPase genes. %%% This project examines the regulation of protein synthesis in chloroplasts by using an experimental system in which an RNA transcript from a specific chloroplast gene is added to an extract from tobacco chloroplasts. The RNA is used as a template for protei n synthesis. This allows features of the RNA that might affect the efficiency or regulation of chloroplast protein synthesis to be studied.

小行星9720444 翻译水平的调控是许多叶绿体基因表达的关键控制点。这种调节通常通过mRNA的5'非翻译区处的RNA-蛋白质相互作用介导。该实验室先前已经表明，特定的RNA-蛋白质相互作用发生在叶绿体编码的ATP合酶基因的5'非翻译区。分析这些相互作用对体外翻译的影响的建议受到了评论者对叶绿体翻译提取物所带来的技术挑战的担忧的阻碍。本研究的目的是通过分析和优化最近报道的制备叶绿体翻译提取物的方案来回答这些问题。该项目有两个主要目标：第一个目标是优化翻译测定，以扩大可以有效翻译的转录本库。两个转录本在体外翻译效率上有几个数量级的差异，将被用作这些实验的底物。顺式作用序列对翻译的影响的分析将使用两种原始底物的嵌合体开始。本研究的第二个目的是扩大提取物制备方案的适用性。将进行实验以将该方案从烟草的叶绿体扩展到其他研究密集型物种的叶绿体，包括菠菜和拟南芥。 该项目将通过POWRE计划得到支持。它将确定一个新的调查线的可行性，应扩大适用性的体外叶绿体翻译调查研究叶绿体编码的ATP酶基因的翻译调控。 本项目通过将特定叶绿体基因的RNA转录物添加到烟草叶绿体提取物中的实验系统，研究叶绿体中蛋白质合成的调节。RNA用作蛋白质合成的模板。这使得可以研究可能影响叶绿体蛋白质合成效率或调节的RNA特征。