Sun E, Wu B W, Tewari K K
Department of Molecular Biology and Biochemistry, School of Biological Sciences, University of California, Irvine 92717.
Mol Cell Biol. 1989 Dec;9(12):5650-9. doi: 10.1128/mcb.9.12.5650-5659.1989.
A cloned pea chloroplast 16S rRNA gene promoter has been characterized in detail by use of a homologous in vitro transcription system that contains a highly purified chloroplast RNA polymerase. The in vivo and in vitro 16S rRNA transcriptional start site has been identified to be a T on the plus strand, 158 bases upstream of the mature 5' end of the gene. BAL 31 deletions of the 16S rRNA leader region demonstrated that the bases between -66 to +30 relative to the transcriptional start site (+1) are necessary for specific 16S transcription. Disruption of canonical TTGACA or TATAAT elements within this region caused complete transcriptional inactivation and prevented protein binding. The topological requirement for 16S transcription was examined by using a construct that synthesized a transcript from the 16S promoter and released it from a pea plastid putative terminator sequence. This minigene was relaxed in vitro with a topoisomerase I from pea chloroplast. It was shown that the 16S promoter was most active when the minigene plasmid was supercoiled.
利用包含高度纯化叶绿体RNA聚合酶的同源体外转录系统，已对克隆的豌豆叶绿体16S rRNA基因启动子进行了详细表征。体内和体外16S rRNA转录起始位点已确定为正链上的一个T，位于该基因成熟5'端上游158个碱基处。对16S rRNA前导区进行BAL 31缺失实验表明，相对于转录起始位点（+1），-66至+30之间的碱基对于16S特异性转录是必需的。该区域内典型的TTGACA或TATAAT元件的破坏导致转录完全失活并阻止蛋白质结合。通过使用一种构建体来检查16S转录的拓扑学要求，该构建体从16S启动子合成转录本并从豌豆质体推定的终止子序列释放它。该小基因在体外用来自豌豆叶绿体的拓扑异构酶I进行松弛处理。结果表明，当小基因质粒为超螺旋时，16S启动子活性最高。
