Abstract
In Sinorhizobium meliloti, the methionine biosynthesis genes metA and metZ are preceded by S-adenosyl-L-methionine (SAM) riboswitches of the SAM-II class. Upon SAM binding, structural changes in the metZ riboswitch were predicted to cause transcriptional termination, generating the sRNA RZ. By contrast, the metA riboswitch was predicted to regulate translation from an AUG1 codon. However, downstream of the metA riboswitch, we found a putative Rho-independent terminator and an in-frame AUG2 codon, which may contribute to metA regulation. We validated the terminator between AUG1 and AUG2, which generates the sRNA RA1 that is processed to RA2. Under high SAM conditions, the activities of the metA and metZ promoters and the steady-state levels of the read-through metA and metZ mRNAs were decreased, while the levels of the RZ and RA2 sRNAs were increased. Under these conditions, the sRNAs and the mRNAs were stabilized. Reporter fusion experiments revealed that the Shine-Dalgarno (SD) sequence in the metA riboswitch is required for translation, which, however, starts 74 nucleotides downstream at AUG2, suggesting a novel translation initiation mechanism. Further, the reporter fusion data supported the following model of RNA-based regulation: Upon SAM binding by the riboswitch, the SD sequence is sequestered to downregulate metA translation, while the mRNA is stabilized. Thus, the SAM-II riboswitches fulfil incoherent, dual regulation, which probably serves to ensure basal metA and metZ mRNA levels under high SAM conditions. This probably helps to adapt to changing conditions and maintain SAM homoeostasis.