Characterization of a natural triple-tandem c-di-GMP riboswitch and application of the riboswitch-based dual-fluorescence reporter
| Autor: | Yang Fu, Shan-Ho Chou, Cao Zheng, Bo Chen, Jianmei Su, Yuqun Xie, Hang Zhou, Jin He, Qing Tang |
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| Jazyk: | angličtina |
| Rok vydání: | 2016 |
| Předmět: |
0301 basic medicine
Riboswitch Untranslated region 030106 microbiology Article 03 medical and health sciences Genes Reporter Escherichia coli RNA Messenger Cyclic GMP Gene Reporter gene Messenger RNA Multidisciplinary Base Sequence biology Computational Biology RNA Gene Expression Regulation Bacterial Molecular biology Cell biology Gene Expression Regulation Cobalamin riboswitch biology.protein Nucleic Acid Conformation Diguanylate cyclase |
| Zdroj: | Scientific Reports |
| ISSN: | 2045-2322 |
| DOI: | 10.1038/srep20871 |
| Popis: | c-di-GMP riboswitches are structured RNAs located in the 5′-untranslated regions (5′-UTRs) of mRNAs that regulate expression of downstream genes in response to changing concentrations of the second messenger c-di-GMP. We discovered three complete c-di-GMP riboswitches (Bc3, Bc4 and Bc5 RNA) with similar structures, which are arranged in tandem to constitute a triple-tandem (Bc3-5 RNA) riboswitch in the 5′-UTR of the cspABCDE mRNA in Bacillus thuringiensis subsp. chinensis CT-43. Our results showed that this natural triple-tandem riboswitch controlled the expression of the reporter gene more stringently and digitally than the double-tandem or single riboswitch. A sandwich-like dual-fluorescence reporter was further constructed by fusing the Bc3-5 RNA gene between the two fluorescence protein genes amcyan and turborfp. This reporter strain was found to exhibit detectable fluorescence color changes under bright field in response to intracellular c-di-GMP level altered by induced expression of diguanylate cyclase (DGC) PleD. Using this system, two putative membrane-bound DGCs from B. thuringiensis and Xanthomonas oryzae were verified to be functional by replacing pleD with the corresponding DGC genes. This report represented the first native triple-tandem riboswitch that was applied to serve as a riboswitch-based dual-fluorescence reporter for the efficient and convenient verification of putative DGC activity in vivo. |
| Databáze: | OpenAIRE |
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