Thyroxine

All riboswitch and promoter sequences are listed in Supplementary Data. Theophylline, fluoride and DNT riboswitches were constructed and inserted into an mRFP1 fluorescent protein expression vector, derived from plasmid pFTV1 (ColE1 origin, Cm^R) (31 (link)). Three theophylline riboswitches (Theo-40, Theo-41 and Theo-45) also controlled the translation of a fusion mRFP1 protein. To create the fusion protein, four or five non-rare codons were introduced between the start codon and SacI restriction site within mRFP1 coding section. All the riboswitches were constructed using standard molecular cloning. Briefly, DNA fragments were computationally designed, synthesized and assembled using either annealing of oligonucleotides, polymerase chain reaction (PCR) assembly of oligonucleotides, or PCR amplification of gBLOCK DNA fragments (Integrated DNA Technologies). DNA fragments were then digested by XbaI and SacI restriction enzymes, followed by ligation with digested plasmid, transformation, plating on selective media and verification of purified plasmid by sequencing. Similarly, promoter replacements were performed by annealing designed pairs of oligonucleotides, followed by digestion with BamHI/XbaI restriction enzymes, ligation, transformation, selective plating and verification by sequencing.
The promoters AEB-3, J23100 and LmrA were selected or designed to significantly vary riboswitch transcription rates (Supplementary Figure S17). The promoter AEB-3 is a result of mutating the −10 and −35 hexamers of promoter J23100, resulting in 10-fold lower transcription rate. The promoter LmrA is a near-consensus promoter with a 5-fold higher transcription rate. Unless noted otherwise in the text, all theophylline and fluoride riboswitches use the J23100 promoter, while all DNT riboswitches used the AEB-3 promoter.
Theophylline and TMR riboswitches were then constructed in plasmids expressing the luciferase reporter protein using standard molecular cloning. The plasmid is derived from the pBESTluc vector (Promega) initially using a pUC19 origin, where the riboswitch-reporter mRNA is transcribed by a Ptac promoter. As described in the text, plasmid origins were replaced with either pBAC, p15A or pFTV1 by PCR amplifying the expression cassette (promoter to transcriptional terminator) and digesting it with BamHI and SpeI, followed by ligation to the corresponding digested vectors, transformation, selective plating and verification by sequencing.
Dopamine and thyroxine riboswitches were constructed in pFTV1-derived plasmids containing the luciferase expression cassette, where pFTV1 was previously modified to insert AatII and HindIII restriction sites after the Ptac promoter and after the start codon, respectively. Riboswitch-encoding DNA fragments were PCR-amplified from designed gBLOCKs, followed by digestion with AatII and HindIII, ligation to digested plasmids, transformation, selective plating and verification by sequencing.