Mechanisms of gene regulation by the conserved small RNA rnTrpL (formerly RcsR1) and its new interaction partners in Sinorhizobium meliloti

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In all investigated organisms, trans-acting small RNAs (sRNAs) regulate gene expression by imperfect base pairing with target mRNAs. Usually more targets are predicted than validated, suggesting that some targets could be addressed under specific conditions. However, examples for sRNA reprogramming in response to stimuli are still lacking. We are investigating the sRNA rnTrpL (formerly RcsR1), which arises due to transcription attenuation of the tryptophan biosynthesis gene trpE(G). This sRNA contains a trpL ORF encoding the leader peptide peTrpL and has many predicted targets. In the first funding period, we validated two of them, the trpDC and rplUrpmA mRNAs, the latter encoding ribosomal proteins L21 and L27. Surprisingly, in contrast to trpDC, rplUrpmA was targeted by rnTrpL in conjunction with the peTrpL peptide and only upon exposure to translation inhibiting antibiotics. The rnTrpL sRNA base pairs with rplU in an antibiotic-dependent ribonucleoprotein (ARNP) complex, which in addition comprises the peTrpL peptide, an antibiotic, and an antibiotic-induced antisense RNA. In the next funding period, we aim to analyze the mechanism by which antibiotics and peTrpL reprogram rnTrpL in favor of rplUrpmA. We will study the assembly and structure of rnTrpL-ARNP complexes, the mechanism and role of rplUrpmA destabilization, and the role of antisense RNAs in this process. Another goal is to identify the rnTrpL interactome under several conditions. The results will uncover the network and mechanisms of a conserved sRNA with specificity change in response to antibiotics.