Lam E, Hanley-Bowdoin L, Chua N H
Laboratory of Plant Molecular Biology, Rockefeller University, New York, New York 10021-6399.
J Biol Chem. 1988 Jun 15;263(17):8288-93.
The large subunit of ribulose 1,5-bisphosphate carboxylase (rbcL) and the beta subunit of chloroplast ATP synthase (atpB) are encoded by divergently transcribed genes on the plastid genome. We have identified DNA binding factors specific for sequences located in the intergenic region between these two genes. Soluble plastid extracts from pea or whole cell extracts from maize protected a maize chloroplast DNA probe containing the 160-base pair region between the 5' ends of rbcL and atpB genes from exonuclease III digestion between positions -16 and -101 relative to the rbcL gene transcription start site. Competition assay with partial sequences from this intergenic region demonstrated that specific sequence(s) are required for the protection. The borders of the binding domain are conserved among the homologous regions of maize, tobacco, spinach, and pea chloroplast genomes. Gel filtration chromatography revealed a molecular weight of about 115,000 for the active complex involved in DNA binding. Using the exonuclease III protection assay, we have also shown that purified Escherichia coli RNA polymerase protects from +25 to -20 of the rbcL gene and from +21 to -23 of the atpB gene relative to their respective transcription start sites. These regions are analogous to open complexes found when E. coli RNA polymerase interacts with the prokaryotic promoters and are consistent with the ability of E. coli RNA polymerase to initiate transcription correctly on linear templates containing these chloroplast promoters. Possible role(s) for the chloroplast DNA binding factor in chloroplast gene expression and its regulation are discussed.
1,5 - 二磷酸核酮糖羧化酶的大亚基（rbcL）和叶绿体ATP合酶的β亚基（atpB）由质体基因组上反向转录的基因编码。我们已经鉴定出了对这两个基因之间基因间隔区序列具有特异性的DNA结合因子。豌豆的可溶性质体提取物或玉米的全细胞提取物能够保护一段玉米叶绿体DNA探针，该探针包含rbcL和atpB基因5'端之间160个碱基对的区域，使其免受核酸外切酶III在相对于rbcL基因转录起始位点-16至-101位置之间的消化。用该基因间隔区的部分序列进行竞争试验表明，保护作用需要特定的序列。结合结构域的边界在玉米、烟草、菠菜和豌豆叶绿体基因组的同源区域中是保守的。凝胶过滤色谱法显示参与DNA结合的活性复合物的分子量约为115,000。使用核酸外切酶III保护试验，我们还表明，纯化的大肠杆菌RNA聚合酶相对于其各自的转录起始位点，能保护rbcL基因从+25至-20以及atpB基因从+21至-23的区域。这些区域类似于大肠杆菌RNA聚合酶与原核启动子相互作用时发现的开放复合物，并且与大肠杆菌RNA聚合酶在含有这些叶绿体启动子的线性模板上正确起始转录的能力一致。本文讨论了叶绿体DNA结合因子在叶绿体基因表达及其调控中的可能作用。
