Optogenetic tools have revolutionized the study of receptor-mediated processes, but such tools are lacking for RNA-controlled systems. In particular, light-activated regulatory RNAs are needed for spatiotemporal control of gene expression. To fill this gap, we used in vitro selection to isolate a novel riboswitch that selectively binds the trans isoform of a stiff-stilbene (amino-tSS)-a rapidly and reversibly photoisomerizing small molecule. Structural probing revealed that the RNA binds amino-tSS about 100-times stronger than the cis photoisoform (amino-cSS). In vitro and in vivo functional analysis showed that the riboswitch, termed Werewolf-1 (Were-1), inhibits translation of a downstream open reading frame when bound to amino-tSS. Photoisomerization of the ligand with a sub-millisecond pulse of light induced the protein expression. In contrast, amino-cSS supported protein expression, which was inhibited upon photoisomerization to amino-tSS. Reversible photoregulation of gene expression using a genetically encoded RNA will likely facilitate high-resolution spatiotemporal analysis of complex RNA processes.

• MeSH
• bakteriální RNA genetika   MeSH
• Escherichia coli genetika   MeSH
• messenger RNA metabolismus   MeSH
• proteosyntéza * MeSH
• regulace genové exprese u bakterií MeSH
• riboswitch * MeSH
• spektrální analýza metody   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH

Nonenzymatic oxidative processes in living organisms are among the inevitable consequences of respiration and environmental conditions. These oxidative processes can lead to the formation of two stereoisomers (R and S) of methionine sulfoxide, and the redox balance between methionine and methionine sulfoxide in proteins has profound implications on their function. Methionine oxidation can be reverted enzymatically by methionine sulfoxide reductases (Msrs). The two enzyme classes known to fulfill this role are MsrA, reducing the (S)-isomer, and MsrB, reducing the (R)-isomer of methionine sulfoxide. They are strictly stereoselective and conserved throughout the tree of life. Under stress conditions such as stationary phase and nutrient starvation, Escherichia coli upregulates the expression of MsrA but a similar effect has not been described for MsrB, raising the conundrum of which pathway enables reduction of the (R)-isomer of methionine sulfoxide in these conditions. Using the recently developed chiral fluorescent probes Sulfox-1, we show that in stationary phase-stressed E. coli, MsrA does have a stereocomplementary activity reducing the (R)-isomer of methionine sulfoxide. However, this activity is not provided by MsrB as expected, but instead by the DMSO reductase complex DmsABC, widely conserved in bacteria. This finding reveals an unexpected diversity in the metabolic enzymes of redox regulation concerning methionine, which should be taken into account in any antibacterial strategies exploiting oxidative stress. DATABASE: The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD013610.

• MeSH
• Escherichia coli enzymologie   MeSH
• fluorescenční barviva chemie   MeSH
• konformace proteinů MeSH
• methionin analogy a deriváty   chemie   metabolismus   MeSH
• methioninsulfoxidreduktasy chemie   metabolismus   MeSH
• oxidace-redukce MeSH
• oxidační stres * MeSH
• oxidoreduktasy chemie   metabolismus   MeSH
• proteiny obsahující železo a síru chemie   metabolismus   MeSH
• proteomika MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH