Kennell J C, Lambowitz A M
Department of Molecular Genetics, Ohio State University, Columbus 43210.
Mol Cell Biol. 1989 Sep;9(9):3603-13. doi: 10.1128/mcb.9.9.3603-3613.1989.
We have developed an in vitro transcription system for Neurospora crassa mitochondrial DNA (mtDNA) and used it to identify transcription initiation sites at the 5' ends of the genes encoding the mitochondrial small and large rRNA and cytochrome b (cob). The in vitro transcription start sites correspond to previously mapped 5' ends of major in vivo transcripts of these genes. Sequences around the three transcription initiation sites define a 15-nucleotide consensus sequence, 5'-TTAGARA(T/G)G(T/G)ARTRR-3', all or part of which appears to be an element of an N. crassa mtDNA promoter. A somewhat looser 11-nucleotide consensus sequence, 5'-TTAGARR(T/G)R(T/G)A-3', was derived by including two additional promoters identified recently. Group I extranuclear mutants, such as [poky] and [SG-3], have a 4-base-pair (bp) deletion in the consensus sequence at the 5' end of the mitochondrial small rRNA and are grossly deficient in mitochondrial small rRNA (R. A. Akins and A. M. Lambowitz, Proc. Natl. Acad. Sci. USA 81:3791-3795, 1984). We show here that the 4-bp deletion in the consensus sequence decreases in vitro transcription from this site by more than 99%. N. crassa mtDNA is similar to Saccharomyces cerevisiae mtDNA in having multiple promoters, including separate promoters for the genes encoding the mitochondrial small and large rRNAs. Our results suggest that the primary effect of the 4-bp deletion in group I extranuclear mutants is to inhibit transcription of the mitochondrial small rRNA, leading to severe deficiency of mitochondrial small rRNA and small ribosomal subunits.
我们开发了一种用于粗糙脉孢菌线粒体DNA（mtDNA）的体外转录系统，并利用它来确定编码线粒体小rRNA、大rRNA和细胞色素b（cob）的基因5'端的转录起始位点。体外转录起始位点与这些基因在体内主要转录本先前定位的5'端相对应。三个转录起始位点周围的序列定义了一个15个核苷酸的共有序列，5'-TTAGARA(T/G)G(T/G)ARTRR-3'，其全部或部分似乎是粗糙脉孢菌mtDNA启动子的一个元件。通过纳入最近鉴定的另外两个启动子，得出了一个稍宽松的11个核苷酸的共有序列，5'-TTAGARR(T/G)R(T/G)A-3'。I类核外突变体，如[poky]和[SG-3]，在线粒体小rRNA 5'端的共有序列中有一个4碱基对（bp）的缺失，并且线粒体小rRNA严重缺乏（R. A. Akins和A. M. Lambowitz，《美国国家科学院院刊》81:3791 - 3795，1984）。我们在此表明，共有序列中的这个bp缺失使该位点的体外转录减少了99%以上。粗糙脉孢菌mtDNA与酿酒酵母mtDNA相似，具有多个启动子，包括分别用于编码线粒体小rRNA和大rRNA的基因的启动子。我们的结果表明，I类核外突变体中4bp缺失的主要作用是抑制线粒体小rRNA的转录，导致线粒体小rRNA和小核糖体亚基严重缺乏。
