Transcription attenuation-derived small RNA rnTrpL regulates tryptophan biosynthesis gene expression in trans

Abstract

Ribosome-mediated transcription attenuation is a basic posttranscriptional regulation mechanism in bacteria. Liberated attenuator RNAs arising in this process are generally considered nonfunctional. In Sinorhizobium meliloti, the tryptophan (Trp) biosynthesis genes are organized into three operons, trpE(G), ppiD-trpDC-moaC-moeA, and trpFBA-accD-folC, of which only the first one, trpE(G), contains a short ORF (trpL) in the 5′-UTR 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. Here, we show that rnTrpL base-pairs with trpD and destabilizes the polycistronic trpDC mRNA, indicating rnTrpL-mediated downregulation of the trpDC operon in trans. Although all three trp operons are regulated in response to Trp availability, only in the two operons trpE(G) and trpDC the Trp-mediated regulation is controlled by rnTrpL. Together, our data show that the trp attenuator coordinates trpE(G) and trpDC expression posttranscriptionally by two fundamentally different mechanisms: ribosome-mediated transcription attenuation in cis and base-pairing in trans. Also, we present evidence that rnTrpL-mediated regulation of trpDC genes expression in trans is conserved in Agrobacterium and Bradyrhizobium, suggesting that the small attenuator RNAs may have additional conserved functions in the control of bacterial gene expression.

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

“…S1). In a previous study, we could show that the sRNA lacZ′-rnTrpL is a functional riboregulator that destabilizes trpDC mRNA (Melior et al, 2019). However, at 10 min and 240 min (4 h) after IPTG addition to cultures of strain 2011 (pSRKGm-rnTrpL), no change in the level of rplUrpmA was detected, although trpC was downregulated, thus confirming the functionality of lacZ′-rnTrpL as a trans -acting sRNA (Fig.…”

“…Each graph contains data from three independent experiments, each performed in duplicates (shown are means and standard deviations). B) Secondary structure of rnTrpL (according to Melior et al, 2019) with indicated mutations used in this work. The rnTrpL derivative with start codon of the trpL sORF exchanged for a stop codon (harbors an inactivated trpL sORF and exhibits the sRNA functions only) was constitutively overproduced from plasmid pRK-rnTrpL-AU1,2UA.…”

“…4A). We performed two-plasmid assays in strain 2011 ΔtrpL using existing lacZ′-rnTrpL derivatives (transcribed from pSRKTc conferring resistance to Tc; Melior et al, 2019) and bicistronic rplUrpmA ′ ::egfp reporter constructs (on pSRKGm conferring resistance to Gm, Fig. S1).…”

“…the small attenuator RNAs and leader peptides, have been considered nonfunctional. Recently, however, we reported that in the soil-dwelling, nitrogen-fixing and Gram-negative plant symbiont S. meliloti , the attenuator RNA rnTrpL is a trans -acting small RNA (sRNA) (Melior et al, 2019). Like in many other bacteria, the trp genes of S. meliloti are organized into several operons, only one of which is regulated by attenuation (Merino et al, 2008).…”

“…Under conditions of Trp availability, fast translation of three consecutive Trp codons in trpL (codons 9 to 11) results in transcription termination. The released sRNA rnTrpL (110 nt) base pairs with and destabilizes trpDC mRNA, thereby posttranscriptionally coordinating the expression of the trpE(G) and trpDC operons (Melior et al, 2019).…”