Rapp W D, Stern D B
Boyce Thompson Institute for Plant Research, Cornell University, Ithaca, NY 14853.
EMBO J. 1992 Mar;11(3):1065-73. doi: 10.1002/j.1460-2075.1992.tb05145.x.
To determine the structure of a functional plant mitochondrial promoter, we have partially purified an RNA polymerase activity that correctly initiates transcription at the maize mitochondrial atp1 promoter in vitro. Using a series of 5' deletion constructs, we found that essential sequences are located within--19 nucleotides (nt) of the transcription initiation site. The region surrounding the initiation site includes conserved sequence motifs previously proposed to be maize mitochondrial promoter elements. Deletion of a conserved 11 nt sequence showed that it is critical for promoter function, but deletion or alteration of conserved upstream G(A/T)3-4 repeats had no effect. When the atp1 11 nt sequence was inserted into different plasmids lacking mitochondrial promoter activity, transcription was only observed for one of these constructs. We infer from these data that the functional promoter extends beyond this motif, most likely in the 5' direction. The maize mitochondrial cox3 and atp6 promoters also direct transcription initiation in this in vitro system, suggesting that it may be widely applicable for studies of mitochondrial transcription in this species.
为了确定功能性植物线粒体启动子的结构，我们部分纯化了一种RNA聚合酶活性，该活性在体外能正确地在玉米线粒体atp1启动子处起始转录。使用一系列5'缺失构建体，我们发现必需序列位于转录起始位点的-19个核苷酸（nt）范围内。起始位点周围的区域包括先前被认为是玉米线粒体启动子元件的保守序列基序。删除一个保守的11 nt序列表明它对启动子功能至关重要，但删除或改变保守的上游G(A/T)3-4重复序列没有影响。当将atp1的11 nt序列插入缺乏线粒体启动子活性的不同质粒中时，仅在其中一个构建体中观察到转录。我们从这些数据推断，功能性启动子延伸超出了这个基序，最有可能是在5'方向。玉米线粒体cox3和atp6启动子在这个体外系统中也指导转录起始，这表明它可能广泛适用于该物种线粒体转录的研究。
