Laufer G, Schaaf G, Bollgönn S, Günzl A
Abteilung Zellbiologie, Zoologisches Institut der Universität Tübingen, D-72076 Tübingen, Germany.
Mol Cell Biol. 1999 Aug;19(8):5466-73. doi: 10.1128/MCB.19.8.5466.
In Trypanosoma brucei, transcription resistant to the mushroom toxin alpha-amanitin is not restricted to the rRNA genes (rDNA), as in higher eukaryotes, but extends to genes encoding the major cell surface proteins variant surface glycoprotein (VSG) and procyclin or procyclic acidic repetitive protein (PARP). Here, we report the development of a homologous cell extract from procyclic T. brucei cells in which rDNA and PARP A and VSG gene promoters drive efficient, accurate, and alpha-amanitin-resistant transcription. A comparative analysis revealed that transcription from the three promoters generally required identical reaction conditions for maximal efficiency. Nevertheless, PARP promoter transcription proved to be exceptional by its high efficiency, its lag phase, a high template DNA concentration optimum, and its tolerance to increasing concentrations of Mn(2+). Mutational analysis for both the PARP and rDNA promoters showed that the proximal and distal core elements were essential for efficient transcription in vitro. Deletion of the upstream control regions (UCRs), however, had a different effect. Whereas PARP UCR deletion reduced transcription efficiency almost 10-fold, deletion of the rDNA UCR had only a minor effect on transcription efficiency.
在布氏锥虫中，对蘑菇毒素α-鹅膏蕈碱具有抗性的转录并不像在高等真核生物中那样仅限于核糖体RNA基因（rDNA），而是扩展到编码主要细胞表面蛋白即变异表面糖蛋白（VSG）和前环素或前环素酸性重复蛋白（PARP）的基因。在此，我们报告了从布氏锥虫前循环期细胞中开发出一种同源细胞提取物，其中rDNA和PARP A以及VSG基因启动子驱动高效、准确且对α-鹅膏蕈碱具有抗性的转录。一项比较分析表明，来自这三种启动子的转录通常需要相同的反应条件才能达到最高效率。然而，PARP启动子转录因其高效率、延迟期、最佳模板DNA高浓度以及对Mn(2+)浓度增加的耐受性而表现出特殊性。对PARP和rDNA启动子的突变分析表明，近端和远端核心元件对于体外高效转录至关重要。然而，上游控制区（UCR）的缺失产生了不同的影响。PARP UCR的缺失使转录效率降低了近10倍，而rDNA UCR的缺失对转录效率只有轻微影响。
