Marczynski G T, Schultz P W, Jaehning J A
Department of Biology, Indiana University, Bloomington 47405.
Mol Cell Biol. 1989 Aug;9(8):3193-202. doi: 10.1128/mcb.9.8.3193-3202.1989.
We have extended an earlier observation that the TATA box for the nuclear GAL10 gene serves as a promoter for the mitochondrial RNA polymerase in in vitro transcription reactions (C. S. Winkley, M. J. Keller, and J. A. Jaehning, J. Biol. Chem. 260:14214-14223, 1985). In this work, we demonstrate that other nuclear genes also have upstream sequences that function in vitro as mitochondrial RNA polymerase promoters. These genes include the GAL7 and MEL1 genes, which are regulated in concert with the GAL10 gene, the sigma repetitive element, and the 2 microns plasmid origin of replication. We used in vitro transcription reactions to test a large number of nuclear DNA sequences that contain critical mitochondrial promoter sequences as defined by Biswas et al. (T. K. Biswas, J. C. Edwards, M. Rabinowitz, and G. S. Getz, J. Biol. Chem. 262:13690-13696, 1987). The results of these experiments allowed us to extend the definition of essential promoter elements. This extended sequence, -ACTATAAACGatcATAG-, was frequently found in the upstream regulatory regions of nuclear genes. On the basis of these observations, we hypothesized that either (i) a catalytic RNA polymerase related to the mitochondrial enzyme functions in the nucleus of the yeast cell or (ii) a DNA sequence recognition factor is shared by the two genetic compartments. By using cells deficient in the catalytic core of the mitochondrial RNA polymerase (rpo41-) and sensitive assays for transcripts initiating from the nuclear promoter sequences, we have conclusively ruled out a role for the catalytic RNA polymerase in synthesizing transcripts from all of the nuclear sequences analyzed. The possibility that a DNA sequence recognition factor functions in both the nucleus and the mitochondria remains to be tested.
我们扩展了之前的一项观察结果，即核GAL10基因的TATA框在体外转录反应中可作为线粒体RNA聚合酶的启动子（C.S.温克利、M.J.凯勒和J.A.杰宁，《生物化学杂志》260:14214 - 14223，1985年）。在这项工作中，我们证明其他核基因也具有在体外作为线粒体RNA聚合酶启动子发挥作用的上游序列。这些基因包括与GAL10基因协同调控的GAL7和MEL1基因、σ重复元件以及2μm质粒复制起点。我们利用体外转录反应来测试大量包含由比斯瓦斯等人定义的关键线粒体启动子序列的核DNA序列（T.K.比斯瓦斯、J.C.爱德华兹、M.拉比诺维茨和G.S.格茨，《生物化学杂志》262:13690 - 13696，1987年）。这些实验结果使我们能够扩展对必需启动子元件的定义。这个扩展后的序列，-ACTATAAACGatcATAG-，在核基因的上游调控区域中经常被发现。基于这些观察结果，我们推测要么（i）一种与线粒体酶相关的催化RNA聚合酶在酵母细胞核中发挥作用，要么（ii）两个遗传区室共享一种DNA序列识别因子。通过使用线粒体RNA聚合酶催化核心缺陷的细胞（rpo41-）以及对从核启动子序列起始的转录本进行灵敏检测，我们已经确凿地排除了催化RNA聚合酶在合成所有分析的核序列转录本中的作用。DNA序列识别因子在细胞核和线粒体中都发挥作用的可能性仍有待检验。
