Jan P S, Stein T, Hehl S, Lisowsky T
Botanisches Institut I, Heinrich-Heine-Universität Düsseldorf, Universitätsstrasse 1, D-40225 Düsseldorf, Germany.
Curr Genet. 1999 Aug;36(1-2):37-48. doi: 10.1007/s002940050470.
The basal mitochondrial transcription apparatus of Saccharomyces cerevisiae consists of the core enzyme for mitochondrial RNA polymerase and the specificity factor. The core enzyme is homologous to those of bacteriophages T3, T7 and SP6 whereas the specificity factor shows similarities with bacterial sigma factors. Recently it was shown that the bacteriophage-type core enzyme is widespread among the eukaryotic lineage and a common picture for the mitochondrial transcription apparatus in eukaryotic cells is now emerging. In contrast to the situation for the core enzyme, the gene for the specificity factor has only been identified from S. cerevisiae and more recently from two other yeast species. As the specificity factor is the key component for initiation of transcription at the mitochondrial promoter we wanted to study in more detail gene expression, regulation, and the function of the promoter of the nuclear MTF1 gene. For this purpose the messenger RNA level for scMTF1 was investigated under a large number of different growth conditions and thereby exhibited a very low, but regulated and carbon source-dependent, expression. Deletion experiments identify the minimal promoter for functional complementation in yeast. To evaluate the functional conservation of the promoter elements the homologous MTF1 gene from the closely related yeast Saccharomyces douglasii was isolated and tested in heterologous complementation experiments. In spite of a highly conserved protein sequence these studies demonstrate that at low-copy number sdMTF1 is not able to substitute for scMTF1 in S. cerevisiae. Promoter exchange experiments with MTF1 from S. cerevisiae and S. douglasii demonstrate that differences in gene expression are responsible for the failure in heterologous complementation. This finding prompted us to compare the promoter regions of MTF1 from four different yeast species. For this purpose the sequences of the 5' regions from S. douglasii, S. kluyveri and Kluyveromyces lactis were determined. A comparison of these sequences identifies significant differences and rapid changes in the intergenic regions, even between closely related yeast species.
酿酒酵母的基础线粒体转录装置由线粒体RNA聚合酶的核心酶和特异性因子组成。核心酶与噬菌体T3、T7和SP6的核心酶同源，而特异性因子与细菌的σ因子有相似之处。最近研究表明，噬菌体类型的核心酶在真核生物谱系中广泛存在，真核细胞线粒体转录装置的常见情况正在显现。与核心酶的情况不同，特异性因子的基因仅在酿酒酵母中被鉴定出来，最近在另外两种酵母物种中也被发现。由于特异性因子是线粒体启动子转录起始的关键成分，我们想更详细地研究核MTF1基因启动子的基因表达、调控及其功能。为此，我们在大量不同的生长条件下研究了scMTF1的信使RNA水平，结果显示其表达水平非常低，但受到调控且依赖于碳源。缺失实验确定了酵母中功能互补的最小启动子。为了评估启动子元件的功能保守性，我们分离了密切相关的酵母道格拉斯酿酒酵母的同源MTF1基因，并在异源互补实验中进行了测试。尽管蛋白质序列高度保守，但这些研究表明，低拷贝数的sdMTF1在酿酒酵母中不能替代scMTF1。酿酒酵母和道格拉斯酿酒酵母的MTF1启动子交换实验表明，基因表达的差异是异源互补失败的原因。这一发现促使我们比较四种不同酵母物种的MTF1启动子区域。为此，我们测定了道格拉斯酿酒酵母、克鲁维酵母和乳酸克鲁维酵母5'区域的序列。这些序列的比较揭示了基因间区域存在显著差异且变化迅速，即使在密切相关的酵母物种之间也是如此。
