Figure 7.

The metA SAM-II riboswitch regulates RNA stability and translation in an opposite manner. A) Schematic representation of the translational fusion plasmid pCD33-egfp with sRNA and CD33-egfp mRNA products, and primer pairs used for qRT-PCR analysis. B) Predicted secondary structure of the riboswitch in its SAM-bound form (compare to Figure. 1B). Shown is the SBPm mutation present in the recombinant sRNAs and CD33-SBPm-egfp mRNA transcribed from pCD33-SBPm-egfp. C) Relative fluorescence of TY and MM cultures of S. meliloti 2011 strains carrying the indicated plasmids. D) Comparison of the CD33-egfp and CD33-SBPm-egfp mRNA levels in TY and MM cultures of S. meliloti 2011 strains harbouring the respective plasmids, shown as log₂FC. The mRNA levels were determined by qRT-PCR using egfp-specific primers (see panel A). E) to H) Before and after rifampicin addition to TY or MM cultures (indicated), RNA was purified from S. meliloti 2011 (panels E and F) or the 2011 ΔRA mutant (panels G and H) harbouring either pCD33-egfp (WT) or pCD33-SBPm-egfp (SBPm). qRT-PCR was performed to determine mRNA half-lives (using egfp-specific primers) or sRNA half-lives (using sRNA-detecting primers; see panel A). Half-life determination of sRNAs with the sRNA-detecting primers is possible, because the cellular sRNA levels are much higher than the mRNA levels (see Figure S7). For further details, see Figure. 1A and Figure. 3. I) Comparison of the relative levels of the CD33-egfp and CD33-SBPm-egfp mRNA in P100 and S100 fractions of S. meliloti 2011 strains, each containing the respective plasmid, using qRT-PCR. The ribosomal protein L27 encoding mRNA rpmA and the dual function sRNA rnTrpL, which harbours the small ORF trpL and is translated as a leaderless small mRNA, were used as controls. All graphs show means and single data points or s.d. of three independent experiments.