POWRE: Optimization and Characterization of Chloroplast Translation In Vitro

9720444翻译水平上的Hollingsworth调控是许多叶绿体基因表达的关键控制点。这种调节通常是通过mRNA 5‘非翻译区的RNA-蛋白质相互作用来介导的。本实验室先前已经证明，特定的RNA-蛋白质相互作用发生在叶绿体编码的ATP合成酶基因的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家对叶绿体翻译提取物存在的技术挑战的担忧的阻碍。这项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分析，以扩大可以有效翻译的转录文库。在体外翻译效率有几个数量级差异的两个转录本将被用作这些实验的底物。分析顺式作用序列对翻译的影响将使用两个原始底物的嵌合体来启动。本研究的第二个目的是扩大协议提取制剂的适用性。将进行实验，将该协议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。它将确定一种新的研究路线的可行性，该方法应将叶绿体体外翻译的适用性扩展到研究叶绿体编码的ATPase基因的翻译调控的研究中。这个项目利用一个实验系统来研究叶绿体中蛋白质合成的调节，在这个实验系统中，特定叶绿体基因的RNA转录物被添加到烟草叶绿体的提取物中。RNA被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。

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.