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

• Applicant: Professorin Dr. Elena Evguenieva-Hackenberg
• Subject Area: Metabolism, Biochemistry and Genetics of Microorganisms
• Term: from 2016 to 2025
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 320729491

• Final Report Year: 2025

Final Report Abstract

RNA-based gene regulation is of great importance for bacterial adaptation to changing environmental conditions. Ribosome-mediated transcription attenuation and mRNA destabilization by trans-acting small RNAs (sRNAs) are two RNA-based mechanisms that are widespread in bacteria. In Sinorhizobium meliloti, the tryptophan (Trp) biosynthesis genes are organized into three operons, trpE(G), trpDC, and trpFBA, of which only the first, trpE(G), is preceded by a short ORF (trpL) and is regulated by transcription attenuation. Under conditions of Trp sufficiency, transcription is terminated between trpL and trpE(G), and a small attenuator RNA, rnTrpL, is produced. We have shown that rnTrpL base-pairs with trpD and destabilizes the trpDC mRNA. Thus, in S. meliloti the trp attenuator coordinates trpE(G) and trpDC expression through two different mechanisms: ribosome-mediated transcription attenuation in cis and base-pairing in trans. Furthermore, we found that in E. coli, where all trp genes are in one operon, the attenuator sRNA rnTrpL can base pair with and destabilize dnaA mRNA. This suggests that in E. coli rnTrpL controls the master regulator of bacterial replication, thereby linking amino acid availability to replication. Additionally, in S. meliloti we observed leader peptide (peTrpL)- and rnTrpL-dependent regulation of the ribosomal operon rplUrpmA, and peTrpL-dependent regulation and the multidrug efflux pump operon smeABR. These operons were regulated by the attenuator products only in the presence of tetracycline. Our results indicated the presence of antibiotic-dependent ribonucleoprotein complexes containing peTrpL and a regulatory sRNA. However, we could not decipher the underlying molecular mechanisms or identify additional rnTrpL targets. During the analysis of the RNA-based response of S. meliloti to tetracycline exposure (used for 10 minutes at subinhibitory concentration), we found that the mRNA levels of more than 300 genes were significantly affected. Our data suggest that this transcriptome remodeling is based on two general mechanisms: mRNA stabilization and premature transcription termination due to decoupling between translation and transcription. Additionally, we found that the gene with the highest induction, which encodes an 83 aa DUF1127 protein, is regulated by a new variant of transcriptional attenuation. Based on our results, we propose an attenuation mechanism that is suitable not only for sensing translation inhibition by antibiotics but also for detecting a shortage of components needed for translation. Our data suggests that the 83 aa DUF1127 protein could be important for survival under conditions of translational deficiency, especially in soil, a habitat with changing nutrient availability, which S. meliloti shares with antibiotic producers.

Publications

• Transcription attenuation-derived small RNA rnTrpL regulates tryptophan biosynthesis gene expression in trans. Nucleic Acids Research, 47(12), 6396-6410.
  Melior, Hendrik; Li, Siqi; Madhugiri, Ramakanth; Stötzel, Maximilian; Azarderakhsh, Saina; Barth-Weber, Susanne; Baumgardt, Kathrin; Ziebuhr, John & Evguenieva-Hackenberg, Elena
  
• Bioinformatic prediction reveals posttranscriptional regulation of the chromosomal replication initiator gene dnaA by the attenuator sRNA rnTrpL in Escherichia coli. RNA Biology, 18(9), 1324-1338.
  Li, Siqi; Edelmann, Daniel; Berghoff, Bork A.; Georg, Jens & Evguenieva-Hackenberg, Elena
  
• The Leader Peptide peTrpL Forms Antibiotic-Containing Ribonucleoprotein Complexes for Posttranscriptional Regulation of Multiresistance Genes. mBio, 11(3).
  Melior, Hendrik; Maaß, Sandra; Li, Siqi; Förstner, Konrad U.; Azarderakhsh, Saina; Varadarajan, Adithi R.; Stötzel, Maximilian; Elhossary, Muhammad; Barth-Weber, Susanne; Ahrens, Christian H.; Becher, Dörte & Evguenieva-Hackenberg, Elena
  
• Reprograming of sRNA target specificity by the leader peptide peTrpL in response to antibiotic exposure. Nucleic Acids Research, 49(5), 2894-2915.
  Melior, Hendrik; Li, Siqi; Stötzel, Maximilian; Maaß, Sandra; Schütz, Rubina; Azarderakhsh, Saina; Shevkoplias, Aleksei; Barth-Weber, Susanne; Baumgardt, Kathrin; Ziebuhr, John; Förstner, Konrad U.; Chervontseva, Zoe; Becher, Dörte & Evguenieva-Hackenberg, Elena
  
• Riboregulation in bacteria: From general principles to novel mechanisms of the trp attenuator and its sRNA and peptide products. WIREs RNA, 13(3).
  Evguenieva‐Hackenberg, Elena
  
• Trans-agierende Attenuator-RNA und Leaderpeptid in Bakterien. BIOspektrum, 27(2), 127–130.
  Melior, Hendrik & Evguenieva-Hackenberg, Elena
  
• Analysis of sRNAs and Their mRNA Targets in Sinorhizobium meliloti: Focus on Half-Life Determination. Methods in Molecular Biology, 239-254. Springer US.
  Scheuer, Robina; Kothe, Jennifer; Wähling, Jan & Evguenieva-Hackenberg, Elena
  
• Early posttranscriptional response to tetracycline exposure in a gram-negative soil bacterium reveals unexpected attenuation mechanism of a DUF1127 gene. openRxiv.
  Kothe, Jennifer A.F.; Sauerwein, Till; Dietz, Theresa; Scheuer, Robina; Elhossary, Muhammad; Barth-Weber, Susanne; Wähling, Jan; Förstner, Konrad U. & Evguenieva-Hackenberg, Elena