Biology subjects

Jagodnik, J.

Publications and source records attributed to Jagodnik, J..

3 recordsLinked to original sources

bioRxiv2025

30S-seq redefines the bacterial Ribosome Binding Site

The translation initiation step is rate limiting for the efficiency of gene expression in all organisms. However, the mechanism of ribosome recruitment to mRNA start sites strikingly differs between eukaryotes and prokaryotes. The eukaryotic small (40S) ribosomal subunit binds 5 end caps and scans for the start codon while the bacterial small (30S) subunit directly binds to the Shine-Dalgarno (SD) motif close to the initiation site. Pioneer studies have shown rare 30S loading events further upstream within 5 untranslated regions (5UTRs), at ribosome standby sites1-3. Together with the frequent occurrence of long bacterial mRNA 5UTRs and degenerated SD sequences, this indicates that the 30S subunit might bind upstream of the SD more commonly than currently thought. We therefore developed 30S-seq to map 30S-mRNA interactions in a bacterial transcriptome (Escherichia coli), inspired by translation complex profile sequencing (TCP-seq) previously used in eukaryotes4,5. Our results provide new and unsuspected insights into the behaviour of 30S and 70S complexes during the canonical translation initiation process. Notably, 30S subunits are recruited upstream of the start codon, primed to receive the SD released by the departing 70S ribosome. Remarkably, we also find hundreds of non-canonical 30S binding sites within mRNA 5UTRs, sometimes over 100 nucleotides upstream of the start region. We validated several of these upstream ribosome binding sites, and demonstrated their strong impact on gene expression. Thus, even in bacteria, ribosomes frequently bind mRNAs outside of the start region to initiate translation, challenging the classic ribosome binding site model.

molecular biology↗

bioRxiv2024

Control of iron acquisition by multiple small RNAs unravels a new role for transcriptional terminator loops in gene regulation

Small RNAs (sRNAs) controlling gene expression by imperfect base-pairing with mRNA(s) are widespread in bacteria and regulate multiple genes, including genes involved in iron homeostasis, through a wide variety of mechanisms. We previously showed that OmrA and OmrB sRNAs repress the synthesis of the Escherichia coli FepA receptor for iron-enterobactin complexes. We now report that five additional sRNAs, namely RprA, RybB, ArrS, RseX and SdsR, that respond to different environmental cues, also repress fepA, independently of one another. While RprA follows the canonical mechanism of pairing with the translation initiation region, repression by ArrS or RseX requires a secondary structure far upstream within the long fepA 5UTR. We also demonstrate a dual action of SdsR, whose 5 end pairs with the fepA translation initiation region while its 3 end behaves like ArrS or RseX. Strikingly, mutation analysis shows a key role for the loops of these sRNAs intrinsic terminators in the regulation. Regulation furthermore depends on both the Hfq chaperone and the RNase E endonuclease. Overall, our data strongly suggest that FepA levels must be tightly controlled under a variety of conditions, and highlight the diversity of mechanisms that underly the regulation of gene expression by sRNAs in bacteria.

microbiology↗

bioRxiv2023

Riboswitch and small RNA modulate btuB translation initiation in Escherichia coli and trigger distinct mRNA regulatory mechanisms

Small RNAs (sRNAs) and riboswitches represent distinct classes of RNA regulators that control gene expression upon sensing metabolic or environmental variations. While sRNAs and riboswitches regulate gene expression by affecting mRNA and protein levels, existing studies have been limited to the characterization of each regulatory system in isolation, suggesting that sRNAs and riboswitches target distinct mRNA populations. We report that the expression of btuB in Escherichia coli, which is regulated by an adenosylcobalamin (AdoCbl) riboswitch, is also controlled by the small RNAs OmrA and, to a lesser extent, OmrB. Strikingly, we find that the riboswitch and sRNAs reduce mRNA levels through distinct pathways. Our data show that while the riboswitch triggers Rho-dependent transcription termination, sRNAs rely on the degradosome to modulate mRNA levels. Importantly, OmrA pairs with the btuB mRNA through its central region, which is not conserved in OmrB, indicating that these two sRNAs may have specific targets in addition to their common regulon. In contrast to canonical sRNA regulation, we find that OmrA repression of btuB is lost using an mRNA binding-deficient Hfq variant. Together, our study demonstrates that riboswitch and sRNAs modulate btuB expression, providing an example of cis- and trans-acting RNA-based regulatory systems maintaining cellular homeostasis.

molecular biology↗