The bacterial leader peptide peTrpL has a conserved function in antibiotic-dependent posttranscriptional regulation of ribosomal genes

Abstract

29The ribosome-dependent attenuator located upstream of bacterial tryptophan biosynthesis genes 30 harbors a small ORF trpL containing tryptophan codons. When tryptophan is available, efficient trpL 31 translation causes transcription termination and release of the attenuator RNA rnTrpL. In Sinorhizobium 32 meliloti, rnTrpL is a trans-acting sRNA. Here, we identified an evolutionary conserved function for the 33 trpL-encoded 14-aa leader peptide peTrpL. Upon exposure to tetracycline, the cellular peTrpL levels 34 were increased and rnTrpL was generated independently of tryptophan availability. Both peTrpL and 35 rnTrpL were found to be involved in tetracycline-dependent destabilization of rplUrpmA mRNA encoding 36 ribosomal proteins L21 and L27. We provide evidence for redirection of the sRNA rnTrpL from its 37 antibiotic-independent target trpDC to rplUrpmA by formation of an antibiotic-dependent 38 ribonucleoprotein complex (ARNP). ARNPs comprising peTrpL, rnTrpL, rplUrpmA and antisense RNA 39 were also observed for other translation-inhibiting antibiotics, suggesting that bacteria evolved 40 mechanisms to utilize antibiotics for mRNA destabilization. 42In addition to the well-studied ribosome-dependent attenuators in Gram-negative bacteria, recently such 83 attenuators were also found to regulate antibiotic resistance operons of Gram-positive bacteria: upon 84 exposure of Bacillus or Listeria to translation-inhibiting antibiotics, ribosome stalling at uORFs prevented 85 transcription termination, thus inducing the expression of downstream resistance genes (Dar et al., 86 2016). The widespread occurrence and high synteny conservation of attenuator uORFs in bacteria 87 raises the question of whether some of these leader peptides may have acquired independent functions 88 in trans during evolution. This hypothesis is supported by the increasing evidence for functional small 89 proteins encoded by sORFs shorter than 50 codons, which often are missing in current genome 90 annotations (Storz et al., 2014). For example, in Drosophila, small proteins of between 11 and 32 aa in 91 length regulate cell morphogenesis (Kondo et al., 2007) and, in Bacillus subtilis, the basic 29-aa protein 92 FbpC was proposed to act as an RNA chaperone (Gaballa et al., 2008), whereas, in E. coli, the 31-aa 93 protein MgtS was shown to interact with two different proteins and regulate Mg 2+ homeostasis (Yin et 94 al., 2018). These few examples illustrate that sORF-encoded proteins have become an important 95 research field (Andrews and Rothnagel, 2014; Storz et al., 2014; Cabrera-Quio et al., 2016; Omasits et 96 al., 2017; Weaver et al., 2019). 97Here, we provide evidence that the trp attenuator-encoded leader peptide peTrpL is involved in the 98 posttranscriptional regulation of the ribosomal genes rplUrpmA in S. meliloti. Analysis of the predicted 99 interaction between the attenuator sRNA rnTrpL and rplUrpmA uncovered that peTrpL and tetracycline 100 (Tc) are both required for an rnTrpL-mediated destabilization of rplUrpmA. Our results ...