Regulation of Photosynthetic Electron Transport Genes in Chlamydomonas Chloroplasts

许多叶绿体基因调控发生在转录后水平。本项目的目的是利用遗传和生化方法对叶绿体翻译起始进行系统的研究。我们将重点关注pet基因，其产物形成细胞色素b6/f复合物，在光系统II和光系统i之间传递电子。预计干扰翻译起始的突变将被引入pet基因，并分析这些突变的第二位点抑制因子。在平行的方法中，将努力通过插入失活来产生核翻译激活子基因的零突变。产生非光合转化体并筛选叶绿体蛋白质积累和翻译缺陷。插入的DNA标记了基因，这有利于克隆突变基因和随后的野生型基因。除了这些遗传方法外，体外生化方法将用于鉴定和表征与5' UTR相互作用的蛋白质。从抑制和插入突变体中提取的蛋白质将被用于检测蛋白质结合因子的差异。该项目的结果将使我们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有更深入的了解。虽然叶绿体有自己的遗传装置，但大部分的调控信号是通过核基因产物的作用传递到叶绿体的。从高等植物和藻类中分离和分析了在这一信号传导过程中存在缺陷的突变体，发现在大多数情况下，叶绿体基因调控发生在转录后水平。此类突变的例子包括影响信使RNA寿命或RNA成熟、蛋白质合成或稳定性以及大分子复合物组装的突变。本项目的两种一般方法中的第一种是将干扰蛋白质合成的突变引入叶绿体基因，然后在核基因中确定补偿抑制突变。同时，将采用生化方法直接检测蛋白因子。在第二种一般方法中，将努力通过将转化DNA引入细胞核来产生破坏核翻译激活因子基因功能的突变。一旦这种突变体被识别出来，“标记”基因就可以被分离出来。拟议的研究结果将导致对控制叶绿体rna翻译的序列以及与这些序列相互作用的蛋白质有更深入的了解。＊＊＊

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. ***