Schaak Janell E, Babitzke Paul, Bevilacqua Philip C
Department of Biochemistry and Molecular Biology, The Huck Institute for Life Sciences, The Pennsylvania State University, University Park, Pennsylvania 16802, USA.
RNA. 2003 Dec;9(12):1502-15. doi: 10.1261/rna.5149603.
Expression of the trpEDCFBA operon of Bacillus subtilis is regulated by transcription attenuation and translation control mechanisms. We recently determined that the B. subtilis trp leader readthrough transcript can adopt a Mg(2+)-dependent tertiary structure that appears to interfere with TRAP-mediated translation control of trpE. In the present study, sequence comparisons to trp leaders from three other Bacillus sp. were made, suggesting that RNA secondary and tertiary structures are phylogenetically conserved. To test this hypothesis, experiments were carried out with the trp leader transcript from Bacillus stearothermophilus. Structure mapping experiments confirmed the predicted secondary structure. Native gel experiments identified a faster mobility species in the presence of Mg(2+), suggesting that a Mg(2+)-dependent tertiary structure forms. Mg(2+)-dependent protection of residues within the first five triplet repeats of the TRAP binding target and a pyrimidine-rich internal loop were observed, consistent with tertiary structure formation between these regions. Structure mapping in the presence of a competitor DNA oligonucleotide allowed the interacting partners to be identified as a single-stranded portion of the purine-rich TRAP binding target and the large downstream pyrimidine-rich internal loop. Thermal denaturation experiments revealed a Mg(2+)- and pH-dependent unfolding transition that was absent for a transcript missing the first five triplet repeats. The stability of several mutant transcripts allowed a large portion of the base-pairing register for the tertiary interaction to be determined. These data indicate that RNA secondary and tertiary structures involved in TRAP-mediated translation control are conserved in at least four Bacillus species.
枯草芽孢杆菌trpEDCFBA操纵子的表达受转录衰减和翻译控制机制调控。我们最近确定，枯草芽孢杆菌trp前导通读转录本可形成一种依赖Mg(2+)的三级结构，该结构似乎会干扰TRAP介导的trpE翻译控制。在本研究中，对来自其他三种芽孢杆菌属细菌的trp前导序列进行了比较，结果表明RNA二级和三级结构在系统发育上是保守的。为验证这一假设，对嗜热脂肪芽孢杆菌的trp前导转录本进行了实验。结构作图实验证实了预测的二级结构。天然凝胶实验发现在存在Mg(2+)的情况下有一种迁移速度更快的物种，表明形成了一种依赖Mg(2+)的三级结构。观察到TRAP结合靶标的前五个三联体重复序列内的残基以及富含嘧啶的内部环存在Mg(2+)依赖性保护，这与这些区域之间形成三级结构一致。在存在竞争DNA寡核苷酸的情况下进行结构作图，确定相互作用的伙伴为富含嘌呤的TRAP结合靶标的单链部分和下游大的富含嘧啶的内部环。热变性实验揭示了一种依赖Mg(2+)和pH的解折叠转变，而缺失前五个三联体重复序列的转录本则没有这种转变。几个突变转录本的稳定性使我们能够确定三级相互作用的大部分碱基配对排列。这些数据表明，参与TRAP介导的翻译控制的RNA二级和三级结构在至少四种芽孢杆菌属细菌中是保守的。
