McDaniel Brooke A, Grundy Frank J, Henkin Tina M
Department of Microbiology, The Ohio State University, Columbus, OH 43210, USA.
Mol Microbiol. 2005 Aug;57(4):1008-21. doi: 10.1111/j.1365-2958.2005.04740.x.
The S box transcription termination control system regulates expression of genes involved in methionine metabolism. Expression of the S box regulon, comprised of 11 transcriptional units in Bacillus subtilis, is induced in response to starvation for methionine. We previously demonstrated that S-adenosylmethionine (SAM) is the molecular effector sensed by the S box leader RNAs during transcription. A secondary structure model for S box leader RNAs was developed based on conservation of primary sequence elements and sequence covariation in helical domains. Covariation of nucleotides in two distantly spaced unpaired regions in the S box leader RNAs suggested that these two domains might interact in the RNA tertiary structure. In this study, site-directed mutagenesis of the covarying residues in two B. subtilis S box leader sequences was employed to test the hypothesis that base-pairing between these regions may be important. The effect of these mutations on in vivo expression, transcription termination in vitro, SAM binding, and leader RNA structure strongly supported the model that interaction between these two regions plays a key role in S box leader function. This provides the first insight into the three-dimensional arrangement of structural elements within the S box RNAs.
S盒转录终止控制系统调节参与甲硫氨酸代谢的基因的表达。枯草芽孢杆菌中由11个转录单元组成的S盒调控子的表达，在甲硫氨酸饥饿时被诱导。我们之前证明，S-腺苷甲硫氨酸（SAM）是转录过程中S盒前导RNA所感知的分子效应物。基于一级序列元件的保守性和螺旋结构域中的序列共变，构建了S盒前导RNA的二级结构模型。S盒前导RNA中两个相距较远的未配对区域的核苷酸共变表明，这两个结构域可能在RNA三级结构中相互作用。在本研究中，通过对两个枯草芽孢杆菌S盒前导序列中共变残基进行定点诱变，来检验这两个区域之间的碱基配对可能很重要这一假说。这些突变对体内表达、体外转录终止、SAM结合和前导RNA结构的影响，有力地支持了这两个区域之间的相互作用在S盒前导功能中起关键作用的模型。这首次揭示了S盒RNA中结构元件的三维排列情况。
