Kandhavelu Meenakshisundaram, Lihavainen Eero, Muthukrishnan Anantha Barathi, Yli-Harja Olli, Ribeiro Andre Sanches
Laboratory of Biosystem Dynamics, Computational Systems Biology Research Group, Department of Signal Processing, Tampere University of Technology, Tampere, Finland.
Biosystems. 2012 Mar;107(3):129-34. doi: 10.1016/j.biosystems.2011.11.001. Epub 2011 Nov 9.
In vitro studies show that the transcriptional dynamics in Escherichia coli is sensitive to Mg(2+) concentration in the cell. We study in vivo how Mg(2+) affects the production of RNA molecules under the control of the lar promoter, P(lar), a lac promoter variant. The target RNA codes for RFP followed by 96 MS2d-GFP binding sites, allowing in vivo detection of individual RNA molecules following transcription. As Mg(2+) concentration is increased, transcripts' production first increases, but then decreases. Results were confirmed by qPCR and gel assay. Analysis of cell to cell diversity in RNA production shows that the variance of RNA numbers changes with Mg(2+). Gel assay confirms changes in the structure of the target RNA. These results suggest that changes in the dynamics of elongation may also affect RNA production, along with changes in the dynamics of the promoter open complex. The findings suggest that changes in metabolite concentration can have multiple, complex effects on the in vivo dynamics of transcription. Comparative analysis of the effects on the dynamics of transcription of other metabolites confirms the significance of the effects of Mg(2+) ions. Namely, we show that Ca(2+) and Fe(2+) have almost negligible effects in comparison to Mg(2+).
体外研究表明，大肠杆菌中的转录动力学对细胞内的Mg(2+)浓度敏感。我们在体内研究了Mg(2+)如何影响在幼虫启动子P(lar)（一种lac启动子变体）控制下的RNA分子的产生。目标RNA编码红色荧光蛋白（RFP），其后跟随96个MS2d - GFP结合位点，这使得在转录后能够在体内检测单个RNA分子。随着Mg(2+)浓度的增加，转录本的产生首先增加，但随后减少。结果通过定量聚合酶链反应（qPCR）和凝胶分析得到证实。对RNA产生过程中细胞间差异的分析表明，RNA数量的方差随Mg(2+)而变化。凝胶分析证实了目标RNA结构的变化。这些结果表明，除了启动子开放复合物动力学的变化外，延伸动力学的变化也可能影响RNA的产生。研究结果表明，代谢物浓度的变化可能对体内转录动力学产生多重、复杂的影响。对其他代谢物对转录动力学影响的比较分析证实了Mg(2+)离子影响的重要性。也就是说，我们表明与Mg(2+)相比，Ca(2+)和Fe(2+)的影响几乎可以忽略不计。
