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首页> 外文期刊>Molecular and Cellular Biology >Development of an in vitro transcription system for Neurospora crassa mitochondrial DNA and identification of transcription initiation sites.
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Development of an in vitro transcription system for Neurospora crassa mitochondrial DNA and identification of transcription initiation sites.

机译：开发克雷索氏菌线粒体DNA体外转录系统并鉴定转录起始位点。

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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.
机译：我们已经开发了一种用于神经孢霉线粒体DNA（mtDNA）的体外转录系统，并用它来识别线粒体大小rRNA和细胞色素b（cob）的编码基因的5'末端的转录起始位点。体外转录起始位点对应于这些基因的主要体内转录本的先前定位的5'末端。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猪笼草的一个元素。 mtDNA启动子。通过包括最近鉴定的两个另外的启动子，得到了一个稍宽松的11个核苷酸的共有序列5'-TTAGARR（T / G）R（T / G）A-3'。第一组I核外突变体，例如[poky]和[SG-3]，在线粒体小rRNA 5'末端的共有序列中有一个4个碱基对（bp）缺失，并且线粒体小rRNA严重缺乏（RA Akins和AM Lambowitz，美国国家科学院学报81：3791-3795，1984）。我们在这里显示，共有序列中的4bp缺失使体外从该位点的转录降低了99％以上。猪笼草mtDNA与啤酒酵母mtDNA相似，具有多个启动子，包括编码线粒体大小rRNA的基因的单独启动子。我们的结果表明，第I组核外突变体中4-bp缺失的主要作用是抑制线粒体小rRNA的转录，从而导致线粒体小rRNA和核糖体小亚基的严重缺乏。

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