Asayama Munehiko, Kato Hideki, Shibato Junko, Shirai Makoto, Ohyama Takashi
Laboratory of Molecular Genetics, School of Agriculture, Ibaraki University, Ami 3-21-1, Inashiki-gun, Ibaraki 300-0332, Japan.
Nucleic Acids Res. 2002 Nov 1;30(21):4658-66. doi: 10.1093/nar/gkf605.
A unique DNA curvature, the CIT, has been found in the 5'-upstream region of the psbA2 gene, which exhibits basal, light-responsive and circadian rhythmic transcription, in a unicellular photosynthetic cyanobacterium, Microcystis aeruginosa K-81. In this study, we report the universality of curvatures found in 5'-upstream regions in the psbA family and the function of the curvature in gene expression. Intrinsic curvatures were identified within 1000 bp upstream from the psbA genes in another cyanobacterium, a red alga and in plants (monocot and dicot). Mutagenized curvatures were constructed and confirmed to have disrupted architecture by gel electrophoresis and atomic force microscopy. Relatively small amounts but light-responsive transcripts of psbA2 were observed in cyanobacterial transformants harboring the mutagenized curvature under light/dark and light/high-light conditions. This shows that the curvature is important for basal transcription. In vitro primer extension and DNA mobility shift assay revealed that factors which might bind to the region upstream from the bending center contribute to the effective basal transcription of psbA2.
在单细胞光合蓝藻铜绿微囊藻K - 81的psbA2基因5'上游区域发现了一种独特的DNA曲率,即CIT,该基因表现出基础转录、光响应转录和昼夜节律转录。在本研究中,我们报告了psbA家族5'上游区域发现的曲率的普遍性以及该曲率在基因表达中的功能。在另一种蓝藻、一种红藻和植物(单子叶植物和双子叶植物)的psbA基因上游1000 bp范围内鉴定出了内在曲率。构建了诱变曲率,并通过凝胶电泳和原子力显微镜证实其结构已被破坏。在光/暗和光/高光条件下,在携带诱变曲率的蓝藻转化体中观察到相对少量但有光响应的psbA2转录本。这表明该曲率对基础转录很重要。体外引物延伸和DNA迁移率变动分析表明,可能与弯曲中心上游区域结合的因子有助于psbA2的有效基础转录。