Plastid RNA Polymerase Sigma Factors

9507329特洛克斯勒RNA聚合酶是叶绿体转录装置的关键组成部分，负责转录、翻译和光合作用所需的约130rRNA、tRNA和蛋白质的表达。4个RNA聚合酶亚基的基因与细菌中RNA聚合酶核心亚单位的编码基因有显著的同源性。叶绿体基因组不包含与细菌中的RNA聚合酶西格玛因子具有明显同源性的蛋白质的基因。这是令人惊讶的，因为许多叶绿体基因包含类似启动子的元件，类似于细菌中Sigma 70型Sigma因子I RNA聚合酶识别的基因中的-10(TATAAT)和-35(TTGACA)共识启动子。缺乏对叶绿体RNA聚合酶中西格玛因子成分的了解是理解叶绿体如何识别其基因的主要差距。这位研究人员通过筛选基因组亚库，从单细胞视紫藻蓝光藻中分离出西格玛因子基因，该亚库含有一个简并的寡核苷酸，编码一个高度保守的区域，在大肠杆菌的西格玛70和枯草芽孢杆菌的西格玛43中称为rpoD盒。推定的西格玛因子的氨基酸序列包含高度保守的结构域，在细菌的西格玛因子中发现了启动子识别、DNA熔化和核心结合。Sigma因子的mRNA是一个2.2kb的多聚腺苷酸化的转录本，仅在光照细胞中表达，这表明该蛋白在核光基因中编码。经限制性内切酶消化后的基因组DNA中含有4-5个与rpoD盒寡核苷酸杂交的限制性片段，这表明铜绿假单胞菌中存在一个叶绿体RNA聚合酶sigma因子家族。这些研究首次在光合作用的真核生物中发现了一种叶绿体RNA聚合酶西格玛因子，并为研究叶绿体基因识别的机制提供了“缺失的环节”。该项目的具体目标是(1)分离和鉴定拟南芥中两个主要的西格玛因子的克隆；(2)研究西格玛因子在黑暗和光照条件下的表达模式。%RNA聚合酶是叶绿体等叶绿体等胞内细胞器转录装置的关键组成部分，负责大约130个基因的表达。这位研究人员最近报告了第一次鉴定出叶绿体聚合酶亚单位的基因，该基因承认了不同类别基因的酶特异性。这种特异性使细胞能够根据环境的变化调整自己的新陈代谢。研究人员对这个亚基的研究应该有助于填补对叶绿体生物学理解的一个重大空白。***

9507329 Troxler RNA polymerase is a key component of the transcriptional apparatus in plastids and is responsible for expression of about 130 genes for rRNAs, tRNAs and proteins required for transcription, translation and photosynthesis. Genes for 4 RNA polymerase subunits have significant homology to genes coding for the core subunits of RNA polymerase in bacteria. Plastid genomes do not contain genes for proteins with recognizable homology to RNA polymerase sigma factors in bacteria. This is surprising because many plastid genes contain promoter-like elements that resemble the -10 (TATAAT) and -35 (TTGACA) consensus promoters in genes recognized by sigma 70 -type sigma factors I RNA polymerase holoenzymes from bacteria. Lack of knowledge of sigma factor components in plastid RNA polymerase is a major gap in understanding how plastids recognize their genes. The investigator has isolate the gene for a sigma factor from the unicellular rhodophyte, Cyanidium caldarium, by screening a genomic sublibrary with a degenerate oligonucleotide coding for a highly conserved region called the "rpoD box" in sigma 70 of Escherichia coli and sigma 43 of Bacillus subtilis. The deduced amino acid sequence of the putative sigma factor contains highly conserved domains for promoter recognition, DNA melting and core binding found in sigma factors from bacteria. The mRNa for the putative sigma factor is a 2.2 kb polyadenylated transcript expressed only in illuminated cells suggesting that this protein is encoded in a nuclear photogene. Genomic DNA digested with restriction enzymes contained 4-5 restriction fragments that hybridized to the rpoD box oligonucleotide suggesting that there is a family of plastid RNA polymerase sigma factors in C. caldarium. These studies are the first to identify a plastid RNA polymerase sigma factor in a photosynthetic eukaryote and provide the "missing link" for investigating mechanisms of plastid gene recognition. The specific aims of its project are to (1) isolate and c haracterize clones for the two principal sigma factors in plastid RNA polymerase from C. caldarium, and (2) examine the expression patterns of sigma factor mRNAs and proteins in dark and light. %%% RNA polymerase is a key component of the transcriptional apparatus in plastid intracellular organelles such as chloroplasts and is responsible for expression of about 130 genes. The investigator has recently reported the first identification of a gene for the plastid polymerase subunit which confess upon the enzyme specificity for different classes of genes. Such specificity enables cells to tailor their metabolism to changes in their environments. The investigator's study of this subunit should help fill a major gap in the understanding of plastid biology. ***