Mulligan R M, Maloney A P, Walbot V
Department of Biological Sciences, Stanford University, CA 94305.
Mol Gen Genet. 1988 Mar;211(3):373-80. doi: 10.1007/BF00425688.
Variation in the length of the 5' non-coding region of mitochondrial gene transcripts could result from multiple transcription initiation sites or post-transcriptional processing events. To distinguish between these possibilities, we have utilized the in vitro capping reaction catalyzed by guanylyl transferase to specifically label the 5' end of primary, unprocessed transcripts. Hybridization of in vitro capped mtRNA to immobilized DNA from the 5' flanking regions of 26 S, 18 S and 5 S rRNA genes and two protein-coding genes, ATP synthase subunit 9 (atp9) and apocytochrome b (cob), identified regions where transcription initiates. Single-strand specific RNase treatment of in vitro capped RNA hybridized to immobilized DNA containing the 5' flanking sequences from cob and atp9 suggests that these genes have multiple transcription initiation sites. Direct mapping of transcription initiation sites for the rRNA genes indicated that single major transcription initiation sites exist at approximately 180 and 230 nucleotides upstream from the mature 26 S and 18 + 5 S rRNA genes, respectively. Labeling of processed transcripts bearing a 5' hydroxyl moiety with T4 polynucleotide kinase and subsequent hybridization to the rRNA genes indicated that the mature forms of the rRNA are processed.
线粒体基因转录本5'非编码区长度的变化可能源于多个转录起始位点或转录后加工事件。为了区分这些可能性，我们利用鸟苷酸转移酶催化的体外加帽反应来特异性标记初级未加工转录本的5'末端。将体外加帽的线粒体RNA与来自26 S、18 S和5 S rRNA基因以及两个蛋白质编码基因（ATP合酶亚基9（atp9）和脱辅基细胞色素b（cob））5'侧翼区域的固定化DNA进行杂交，确定了转录起始的区域。对与含有cob和atp9 5'侧翼序列的固定化DNA杂交的体外加帽RNA进行单链特异性核糖核酸酶处理，表明这些基因有多个转录起始位点。对rRNA基因转录起始位点的直接定位表明，在成熟的26 S和18 + 5 S rRNA基因上游约180和230个核苷酸处分别存在单个主要转录起始位点。用T4多核苷酸激酶对带有5'羟基部分的加工转录本进行标记，随后与rRNA基因杂交，表明rRNA的成熟形式是经过加工的。
