Pinkham J L, Dudley A M, Mason T L
Department of Biochemistry and Molecular Biology, University of Massachusetts, Amherst 01003-4505.
Mol Cell Biol. 1994 Jul;14(7):4643-52. doi: 10.1128/mcb.14.7.4643-4652.1994.
An in vivo expression system has been developed for controlling the transcription of individual genes in the mitochondrial genome of Saccharomyces cerevisiae. The bacteriophage T7 RNA polymerase (T7Pol), fused to the COXIV mitchondrial import peptide and expressed under the control of either the GAL1 or the ADH1 promoter, efficiently transcribes a target gene, T7-COX2, in the mitochondrial genome. Cells bearing the T7-COX2 gene, but lacking wild-type COX2, require T7Pol for respiration. Functional expression of T7-COX2 is completely dependent on the COX2-specific translational activator Pet111p, despite additional nucleotides at the 5' end of the T7-COX2 transcript. Expression of mitochondrion-targeted T7Pol at high levels from the GAL1 promoter has no detectable effect on mitochondrial function in rho+ cells lacking the T7-COX2 target gene, but in cells with T7-COX2 integrated into the mitochondrial genome, an equivalent level of T7Pol expression causes severe respiratory deficiency. In comparison with wild-type COX2 expression, steady-state levels of T7-COX2 mRNA increase fivefold when transcription is driven by T7Pol expressed from the ADH1 promoter, yet COXII protein levels and cellular respiration rates decrease by about 50%. This discoordinate expression of mRNA and protein provides additional evidence for posttranscriptional control of COX2 expression.
已开发出一种体内表达系统，用于控制酿酒酵母线粒体基因组中单个基因的转录。与细胞色素c氧化酶亚基IV（COXIV）线粒体导入肽融合并在GAL1或ADH1启动子控制下表达的噬菌体T7 RNA聚合酶（T7Pol），能有效地转录线粒体基因组中的靶基因T7-COX2。携带T7-COX2基因但缺乏野生型COX2的细胞，呼吸需要T7Pol。尽管T7-COX2转录本的5'端有额外的核苷酸，但T7-COX2的功能性表达完全依赖于COX2特异性翻译激活因子Pet111p。在缺乏T7-COX2靶基因的rho+细胞中，从GAL1启动子高水平表达靶向线粒体的T7Pol对线粒体功能没有可检测到的影响，但在T7-COX2整合到线粒体基因组的细胞中，同等水平的T7Pol表达会导致严重的呼吸缺陷。与野生型COX2表达相比，当由ADH1启动子表达的T7Pol驱动转录时，T7-COX2 mRNA的稳态水平增加五倍，但COXII蛋白水平和细胞呼吸速率下降约50%。mRNA和蛋白质的这种不协调表达为COX2表达的转录后控制提供了额外的证据。
