In the absence of extensive transcription control mechanisms the pathogenic parasite Trypanosoma brucei crucially depends on translation regulation to orchestrate gene expression. However, molecular insight into regulating protein biosynthesis is sparse. Here we analyze the small non-coding RNA (ncRNA) interactome of ribosomes in T. brucei during different growth conditions and life stages. Ribosome-associated ncRNAs have recently been recognized as unprecedented regulators of ribosome functions. Our data show that the tRNAThr 3´half is produced during nutrient deprivation and becomes one of the most abundant tRNA-derived RNA fragments (tdRs). tRNAThr halves associate with ribosomes and polysomes and stimulate translation by facilitating mRNA loading during stress recovery once starvation conditions ceased. Blocking or depleting the endogenous tRNAThr halves mitigates this stimulatory effect both in vivo and in vitro. T. brucei and its close relatives lack the well-described mammalian enzymes for tRNA half processing, thus hinting at a unique tdR biogenesis in these parasites.

Ever since the “great oxidation event,” Earth’s cellular life forms had to cope with the danger of reactive oxygen species (ROS) affecting the integrity of biomolecules and hampering cellular metabolism circuits. Consequently, increasing ROS levels in the biosphere represented growing stress levels and thus shaped the evolution of species. Whether the ROS were produced endogenously or exogenously, different systems evolved to remove the ROS and repair the damage they inflicted. If ROS outweigh the cell’s capacity to remove the threat, we speak of oxidative stress. The injuries through oxidative stress in cells are diverse. This article reviews the damage oxidative stress imposes on the different steps of the central dogma of molecular biology in bacteria, focusing in particular on the RNA machines involved in transcription and translation.

Almost two-thirds of the microbiome’s biomass has been predicted to be in a non-proliferating, and thus dormant, growth state. It is assumed that dormancy goes hand in hand with global downregulation of gene expression. However, it remains largely unknown how bacteria manage to establish this resting phenotype at the molecular level. Recently small non-protein-coding RNAs (sRNAs or ncRNAs) have been suggested to be involved in establishing the non-proliferating state in bacteria. Here, we have deep sequenced the small transcriptome of Escherichia coli in the exponential and stationary phases and analyzed the resulting reads by a novel biocomputational pipeline STARPA (Stable RNA Processing Product Analyzer). Our analysis reveals over 12,000 small transcripts enriched during both growth stages. Differential expression analysis reveals distinct sRNAs enriched in the stationary phase that originate from various genomic regions, including transfer RNA (tRNA) fragments. Furthermore, expression profiling by Northern blot and RT-qPCR analyses confirms the growth phase-dependent expression of several enriched sRNAs. Our study adds to the existing repertoire of bacterial sRNAs and suggests a role for some of these small molecules in establishing and maintaining stationary phase as well as the bacterial stress response. Functional characterization of these detected sRNAs has the potential of unraveling novel regulatory networks central for stationary phase biology.

Post-transcriptional modifications are added to ribosomal RNAs (rRNAs) to govern ribosome biogenesis and to fine-tune protein biosynthesis. In Escherichia coli and related bacteria, RlhA uniquely catalyzes formation of a 5-hydroxycytidine (ho5C) at position 2501 of 23S rRNA. However, the molecular and biological functions as well as the regulation of ho5C2501 modification remain unclear. We measured growth curves with the modification-deficient ΔrlhA strain and quantified the extent of the modification during different conditions by mass spectrometry and reverse transcription. The levels of ho5C2501 in E. coli ribosomes turned out to be highly dynamic and growth phase-dependent, with the most effective hydroxylation yields observed in the stationary phase. We demonstrated a direct effect of ho5C2501 on translation efficiencies in vitro and in vivo. High ho5C2501 levels reduced protein biosynthesis which however turned out to be beneficial for E. coli for adapting to oxidative stress. This functional advantage was small under optimal conditions or during heat or cold shock, but becomes pronounced in the presence of hydrogen peroxide. Taken together, these data provided first functional insights into the role of this unique 23S rRNA modification for ribosome functions and bacterial growth under oxidative stress.