Regulation of Photosynthetic Electron Transport Genes in Chlamydomonas Chloroplasts

摘要叶绿体基因调控大多发生在转录后水平. 本项目的目的是利用遗传学和生物化学方法对叶绿体翻译起始进行系统的研究。 我们将集中于pet基因，其产物形成细胞色素b6/f复合物，该复合物在光系统II和光系统I之间传输电子。 将预测干扰翻译起始的突变引入pet基因，并分析这些突变的第二位点抑制子。 在平行的方法中，将努力通过插入失活在核翻译激活因子基因中产生无效突变。 将产生非光合转化体并筛选叶绿体蛋白质积累和翻译的缺陷。 插入的DNA标记基因，这有助于克隆突变基因，随后克隆野生型基因。 除了这些遗传方法之外，体外生物化学方法将用于鉴定和表征与5' UTR相互作用的蛋白质。 将测定抑制突变体和插入突变体的蛋白提取物中蛋白结合因子的任何差异。 该项目的结果将导致对控制翻译的顺式作用序列以及与这些序列相互作用的蛋白质的更好理解。 虽然叶绿体有自己的遗传器，但大多数调节信号是通过核基因产物的作用传递到叶绿体的。 在高等植物和藻类的突变体的分离和分析，是在这个信号传导过程中有缺陷的，在大多数情况下，叶绿体基因调控发生在转录后水平。 此类突变的实例包括影响信使RNA寿命或RNA成熟、蛋白质合成或稳定性以及大分子复合物的组装的那些。 在这个项目中的两个一般方法中的第一个是将干扰蛋白质合成的突变引入叶绿体基因，然后在核基因中鉴定补偿抑制突变。 同时，采用生物化学方法对蛋白质因子进行直接检测。 在第二种一般方法中，将努力通过将转化DNA引入细胞核中来产生破坏核翻译激活因子基因功能的突变。 一旦鉴定出这样的突变体，就可以分离出“标记的”基因。 拟议的研究结果将导致更好地了解控制叶绿体RNA翻译的序列，以及与这些序列相互作用的蛋白质。 ***

Abstract 9406550 Much of chloroplast gene regulation occurs at the post- transcriptional level. The aim of this project is to undertake a systematic study of chloroplast translation initiation using genetic and biochemical approaches. We will focus on the pet genes, whose products form the cytochrome b6/f complex, which transports electrons between photosystem II and photosystem I. Mutations predicted to interfere with translation initiation will be introduced into pet genes and second-site suppressors of these mutations will be analyzed. In a parallel approach, efforts will be made to generate null mutations in nuclear translational activator genes by insertional inactivation. Non-photosyntheic transformants will be generated and screened for defects in chloroplast protein accumulation and translation. The inserted DNA tags the gene, which facilitates cloning the mutated gene and subsequently the wild-type version. In addition to these genetic approaches, in vitro biochemical methods will be used to identify and characterize proteins that interact with the 5' UTR. Protein extracts from suppressor and insertion mutants will be assayed for any differences in protein binding factors. The results of the project will lead to a greater understanding of the cis-acting sequences controlling translation, and the proteins that interact with these sequences. %%% Although chloroplasts contain their own genetic apparatus, most of the regulatory signals are transmitted to the chloroplast by the action of nuclear gene products. The isolation and analysis of mutants in both higher plants and algae that are defective in this signaling process have revealed that in most cases, chloroplast gene regulation occurs at the post-transcriptional level. Examples of such mutations include those that affect messenger RNA lifetimes or RNA maturation, protein synthesis or stability, and the assembly of macromolecular complexes. The first of two general approaches in this projec t is to introduce mutations that interfere with protein synthesis into chloroplast genes and then to identify compensating suppressor mutations in nuclear genes. At the same time, biochemical methods will be used to detect the protein factors directly. In a second general approach, efforts will be made to generate mutations that disrupt the functions of nuclear translational activator genes, by introducing transforming DNA into the nucleus. Once such mutants have been identified, the "tagged" gene can be isolated. The results of the proposed research will lead to a greater understanding of the sequences that control translation of chloroplast RNAs, and of the proteins that interact with these sequences. ***