FIG 6.

Cdi2_4 is a transcriptional c-di-GMP-II riboswitch. (A) Predicted Cdi2_4 c-di-GMP-II riboswitch-mediated transcription regulation. c-di-GMP is predicted to increase transcription past a predicted Rho-independent transcription terminator. Upon c-di-GMP binding to the aptamer, the P1 stem folds and sequesters nucleotides needed for the terminator formation and therefore acts as an antiterminator. Positions 231 and 146 correspond to the last nucleotides of the full-length transcript and the predicted terminated transcript, respectively. GC nucleotides (underlined) were added to the template for transcription priming using GC dinucleotides. The predicted Cdi2_4 aptamer is illustrated based on its predicted secondary structure (25). The transcription terminator folding was predicted using the RNAfold web server (http://rna.tbi.univie.ac.at/cgi-bin/RNAfold.cgi) (62). (B) Single-round in vitro transcription of the Cdi2_4 riboswitch template. Transcription reactions were carried out as a function of c-di-GMP concentration. Read-through (RT) and terminated (T) products are indicated on the right. Transcripts of 231 nt (RT) and 146 nt (T) were labeled and used as molecular size makers (MWM). The reactions were done in duplicate, and a representative image is shown. (C) The percentages of read-through products are plotted as a function of c-di-GMP concentration as quantified from panel B. The plot shows a two-state model from which a T₅₀ value of 70 ± 12 nM c-di-GMP was calculated.