Biswas T K
Department of Pathology, University of Chicago, 5841 S. Maryland Avenue, Chicago, IL 60637, USA.
Gene. 1998 Jun 8;212(2):305-14. doi: 10.1016/s0378-1119(98)00133-4.
Prior work has demonstrated that a conserved nonanucleotide [5'-TATAAGTAA(+2)] promoter sequence is used by the mitochondrial [mt]1 RNA polymerase in Saccharomyces cerevisiae. However, the highly AT-rich yeast mt genome carries many other promoter-like sequences, but only a fraction of them are involved in gene-specific transcription. To examine the sequence variability of this nonanucleotide promoter motif, single or multiple nt substitutions were introduced into the canonical promoter sequence. The transcriptional activity of these altered promoter sequences was examined under the in-vitro reaction conditions. The results presented here determined that several variant promoter sequences (i. e. TAAAAGTAA, TATAAGAAA, TATAAGTAG, TATAAGAAG, TATAAGAGA, TATAAGGGA, TATAAGTGG, TAAAAGTAG) were efficiently used by the mtRNA polymerase. However, a single (i.e. AATAAGTAA, TTTAAGTAA, TATTAGTAA, TATAACTAA, TATAAGGAA, TATAAGTAT) or multiple (TATAGGAAA, TAAAAGGAA, TATAGGGAA, TAAAGGAAA, TAAAGGGAA) nt substitution(s) in other locations drastically reduced mt promoter function. Interestingly, some of these poorly or partially active promoter variants (i.e. TATAAGGAA, TATAAGTAT, TATAAGTCA) became fully functional in the presence of sequence-specific dinucleotide primer. Since dinucleotide primer bypasses the first phosphodiester bond formation in transcription, it is suggested that the -1T-->G, +1A-->C and +2A-->T mutations affect mt transcription at the level of initiation rather than polymerase binding.
先前的研究表明，酿酒酵母中的线粒体[mt]1 RNA聚合酶使用保守的九核苷酸[5'-TATAAGTAA(+2)]启动子序列。然而，富含AT的酵母线粒体基因组携带许多其他类似启动子的序列，但其中只有一部分参与基因特异性转录。为了研究这种九核苷酸启动子基序的序列变异性，将单个或多个核苷酸替换引入到标准启动子序列中。在体外反应条件下检测这些改变后的启动子序列的转录活性。此处给出的结果表明，几种变体启动子序列（即TAAAAGTAA、TATAAGAAA、TATAAGTAG、TATAAGAAG、TATAAGAGA、TATAAGGGA、TATAAGTGG、TAAAAGTAG）能被mtRNA聚合酶有效利用。然而，在其他位置的单个（即AATAAGTAA、TTTAAGTAA、TATTAGTAA、TATAACTAA、TATAAGGAA、TATAAGTAT）或多个（TATAGGAAA、TAAAAGGAA、TATAGGGAA、TAAAGGAAA、TAAAGGGAA）核苷酸替换会大幅降低线粒体启动子功能。有趣的是，这些活性较差或部分活性的启动子变体中的一些（即TATAAGGAA、TATAAGTAT、TATAAGTCA）在存在序列特异性二核苷酸引物的情况下变得完全有功能。由于二核苷酸引物绕过了转录中第一个磷酸二酯键的形成，因此表明-1T→G、+1A→C和+2A→T突变在起始水平而非聚合酶结合水平影响线粒体转录。
