Projects

Research topics

We are investigating RNA level regulation in bacteria, using Sinorhizobium meliloti—a soil dwelling, nitrogen fixing plant symbiont—as a model system. Current projects focus on upstream open reading frames (uORFs) and upstream ribosome binding sites (uRBSs), as well as on small genes

Genome-wide prediction of uRBSs and the regulatory “sitting turtle” ribosome

We hypothesized that upstream ribosome binding sites (uRBSs) and upstream open reading frames (uORFs), both of which modulate gene expression, might be more common in bacteria than previously appreciated. Accordingly, we performed genome wide predictions of uRBSs and uORFs in Sinorhizobium meliloti and examined the resulting candidates. Unexpectedly, we identified several uRBSs that were ribosome occupied according to Hadjeras et al. (2023) yet were almost silent in eGFP translational fusion assays. These sites were positioned close to the downstream main ribosome binding site (mRBS). We propose that such uRBSs mediate ribosomal occlusion, thereby down regulating the downstream open reading frame, and we term the ribosomes bound to these sites “sitting turtle” ribosomes. (Dietz, Hahnfeld et al., 2026.; doi: 10.64898/2026.05.12.723736)

Different classes of uRBSs and corresponding uORFs were identified in S. meliloti (Dietz, Hahnfeld et al., 2026). We are currently analyzing additional predicted uRBSs

Antibiotic exposure post-transcriptionally activates a duf1127 gene

S. meliloti shares its free living habitat with antibiotic producing microorganisms; consequently, we performed RNA seq to examine its early transcriptional response to sub inhibitory tetracycline exposure. The most strongly induced transcript encoded a DUF1127 protein. We discovered that this gene is regulated by transcriptional attenuation that depends on the translation of an upstream open reading frame (uORF). Attenuation is governed either by impaired translation initiation or—in the absence of ribosome binding—by base pairing between the upstream ribosome binding site (uRBS) and C rich codons of the uORF; the latter are probably a Rho-binding site (Kothe et al., 2025). These findings suggest that S. meliloti uses this mechanism to sense both ribosome availability and translation efficiency. Building on these results, future work will help elucidate the functional role of this DUF1127 protein and its closest homologs

Model of the novel attenuation mechanism controlling duf11271 expression Our data suggest that two sequence elements (marked in red) are needed for transcriptional attenuation of duf11271 in the absence of tetracycline (Tc). The first element is located at codons 3 to 10 of the uORF and is accessible under conditions of normal translation, while the second element is located downstream of the uORF. A) For successful attenuation in the absence of Tc, a ribosome must translate at least the first 14 uORF codons (translation termination at codon 15 still allows for attenuation). In the time window before the next ribosome occupies the ribosome binding site (RBS) of the uORF, the translated attenuation element is accessible for a factor (presumably the Rho hexamer), which then also binds the second attenuation element in the nascent RNA and leads to premature transcriptional termination. B) In the presence of Tc, the translation initiation at the start codon of the uORF is impaired. The stalled ribosome blocks the accessibility to the first attenuation element and therefore transcription proceeds into duf11271 (attenuation is relieved). C) According to our data, a delay in the occupancy of the uORF RBS by a pioneering ribosome would also lead to relieve of attenuation and duf11271 transcription, because the nascent RNA forms a secondary structure that blocks the first attenuation element. From Kothe et al. (2025) doi: 10.1080/15476286.2025.2521887

The small proteome in S. meliloti

Small proteins are frequently omitted from genome annotations, although they can play critical biological roles. Recently, in collaboration with others (Hadjeras et al., 2023), we identified novel small ORFs (sORFs) in S. meliloti using ribosome profiling combined with mass spectrometry. The resulting Ribo seq data can be accessed via JBrowse (http://www.bioinf.uni-freiburg.de/ribobase)

Detection of small ORFs and small proteins in Sinorhizobium meliloti From Hadjeras et al. (2023), doi: 10.1093/femsml/uqad012

A SAM-II riboswitch controls ribosome access to an upstream ribosome binding site (uRBS)

Riboswitches are cis acting RNA elements that bind specific metabolites and modulate the expression of downstream genes. SAM riboswitches bind S adenosyl L methionine (SAM), the principal methyl donor in cellular biochemistry. We discovered two unexpected roles for a SAM II riboswitch that regulates metA in S. meliloti: in the SAM bound state it down regulates metA translation via a novel mechanism, and it also stabilizes the mRNA. The translational control involves an upstream ribosome binding site (uRBS). These dual mechanisms likely help the cell adapt to fluctuating conditions and maintain SAM homeostasis (Scheuer et al., 2022)

Model of metA regulation On the left: Under low SAM conditions, transcription from the metA promoter (PmetA) is increased and the riboswitch is in the SAM-free state. The Shine-Dalgarno (SD) sequence downstream of the riboswitch aptamer is accessible and activates translation in the read-through mRNA from the starting codon AUG2. Under these conditions, metA mRNA is destabilized. On the right: Under high SAM conditions, transcription from PmetA is decreased, the riboswitch is in the SAM-bound state, the SD controlled by the riboswitch is not accessible and metA translation is repressed. Under these conditions, metA mRNA is stabilized. From Scheuer et al. (2002), doi: 10.1080/15476286.2022.2110380

References

1.    Dietz T, Hahnfeld JM, Neumann S, Reinsch YA, Wenz T, Barth-Weber S, Blom J, Goesmann A, Evguenieva-Hackenberg E. Predicted Bacterial uRBSs Reveal Translational Coupling and Ribosome Mediated RBS Occlusion as Gene-Controlling Mechanisms. bioRxiv 2026 doi: https://doi.org/10.64898/2026.05.12.723736; accepted by microLife.
2.    Hadjeras L, Heiniger B, Maaß S, Scheuer R, Gelhausen R, Azarderakhsh S, Barth-Weber S, Backofen R, Becher D, Ahrens CH, Sharma CM, Evguenieva-Hackenberg E. (2023) Unraveling the small proteome of the plant symbiont Sinorhizobium meliloti by ribosome profiling and proteogenomics. microLife 4:uqad012. 
3.    Kothe JAF, Sauerwein T, Dietz T, Scheuer R, Elhossary M, Barth-Weber S, Wähling J, Förstner KU, Evguenieva-Hackenberg E. (2025) Early posttranscriptional response to tetracycline exposure in a gram-negative soil bacterium reveals unexpected attenuation mechanism of a DUF1127 gene. RNA Biol. 22:1-16.
4.    Scheuer R, Dietz T, Kretz J, Hadjeras L, McIntosh M, Evguenieva-Hackenberg E. (2022) Incoherent dual regulation by a SAM-II riboswitch controlling translation at a distance. RNA Biol. 19:980-995.