Reprograming of sRNA target specificity by the leader peptide peTrpL in response to antibiotic exposure
Hendrik Melior, Siqi Li, Maximilian Stötzel, Sandra Maaß, Rubina Schütz, Saina Azarderakhsh, Aleksei Shevkoplias, Susanne Barth-Weber, Kathrin Baumgardt, John Ziebuhr, Konrad U Förstner, Zoe Chervontseva, Dörte Becher, Elena Evguenieva-Hackenberg
Trans-acting regulatory RNAs have the capacity to base pair with more mRNAs than generally detected under defined conditions, raising the possibility that sRNA target specificities vary depending on the specific metabolic or environmental conditions. In Sinorhizobium meliloti, the sRNA rnTrpL is derived from a tryptophan (Trp) transcription attenuator located upstream of the Trp biosynthesis gene trpE(G). The sRNA rnTrpL contains a small ORF, trpL, encoding the 14-aa leader peptide peTrpL. If Trp is available, efficient trpL translation causes transcription termination and liberation of rnTrpL, which subsequently acts to downregulate the trpDC operon, while peTrpL is known to have a Trp-independent role in posttranscriptional regulation of antibiotic resistance mechanisms. Here, we show that tetracycline (Tc) causes rnTrpL accumulation independently of Trp availability. In the presence of Tc, rnTrpL and peTrpL act collectively to destabilize rplUrpmA mRNA encoding ribosomal proteins L21 and L27. The three molecules, rnTrpL, peTrpL, and rplUrpmA mRNA, form an antibiotic-dependent ribonucleoprotein complex (ARNP). In vitro reconstitution of this ARNP in the presence of competing trpD and rplU transcripts revealed that peTrpL and Tc cause a shift of rnTrpL specificity towards rplU, suggesting that sRNA target prioritization may be readjusted in response to changing environmental conditions.
Trans-agierende Attenuator-RNA und Leaderpeptid in Bakterien
Hendrik Melior and Elena Evguenieva-Hackenberg
Bacterial transcription attenuators are a source of small RNAs (sRNAs) and leader peptides, for which no own functions were known. How-ever, the attenuator sRNA of the tryptophan (Trp) biosynthesis operon regulates gene expression in trans according to the Trp-availability. Moreover, the cognate leader peptide adopted Trp-independent functions. It builds antibiotic-dependent ribonucleoprotein complexes (ARNPs) for sRNA reprogramming and regulation of ribosomal and multiresistance genes.
High complexity of Glutamine synthetase regulation in Methanosarcina mazei: Small protein 26 interacts and enhances glutamine synthetase activity
Miriam Gutt, Britta Jordan, Katrin Weidenbach, Mirja Gudzuhn, Claudia Kiessling, Liam Cassidy, Andreas Helbig, Andreas Tholey, Dennis Joshua Pyper, Nina Kubatova, Harald Schwalbe and Ruth Anne Schmitz
Small ORF (sORF)‐encoded small proteins have been overlooked for a long time due to challenges in prediction and distinguishing between coding‐ and noncoding‐predicted sORFs and in their biochemical detection and characterization. We report on the first biochemical and functional characterization of a small protein (sP26) in the archaeal model organism Methanosarcina mazei, comprising 23 amino acids. The corresponding encoding leaderless mRNA (spRNA26) is highly conserved on nucleotide level as well as on the coded amino acids within numerous Methanosarcina strains strongly arguing for a cellular function of the small protein. spRNA26 level is significantly enhanced under nitrogen limitation, but also under oxygen and salt stress conditions. Using heterologously expressed and purified sP26 in independent biochemical approaches [pull‐down by affinity chromatography followed by MS analysis, reverse pull‐down, microscale thermophoresis, size‐exclusion chromatography, and nuclear magnetic resonance spectroscopy (NMR) analysis], we observed that sP26 interacts and forms complexes with M. mazei glutamine synthetase (GlnA1) with high affinity (app. KD = 0.76 µm ± 0.29 µm). Moreover, seven amino acids were identified by NMR analysis to directly interact with GlnA1. Upon interaction with sP26, GlnA1 activity is significantly stimulated, independently and in addition to the known activation by the metabolite 2‐oxoglutarate (2‐OG). Besides, strong interaction of sP26 with the PII‐like protein GlnK1 was demonstrated (app. KD = 2.9 µm ± 0.9 µm). On the basis of these findings, we propose that in addition to 2‐OG, sP26 enhances GlnA1 activity under nitrogen limitation most likely by stabilizing the dodecameric structure of GlnA1.