Selective translation of leaderless mRNAs by specialized ribosomes generated by MazF in Escherichia coli.

Escherichia coli (E. coli) mazEF is a stress-induced toxin-antitoxin (TA) module. The toxin MazF is an endoribonuclease that cleaves single-stranded mRNAs at ACA sequences. Here, we show that MazF cleaves at ACA sites at or closely upstream of the AUG start codon of some specific mRNAs and thereby generates leaderless mRNAs. Moreover, we provide evidence that MazF also targets 16S rRNA within 30S ribosomal subunits at the decoding center, thereby removing 43 nucleotides from the 3' terminus. As this region comprises the anti-Shine-Dalgarno (aSD) sequence that is required for translation initiation on canonical mRNAs, a subpopulation of ribosomes is formed that selectively translates the described leaderless mRNAs both in vivo and in vitro. Thus, we have discovered a modified translation machinery that is generated in response to MazF induction and that probably serves for stress adaptation in Escherichia coli.

Autoregulation of mazEF expression underlies growth heterogeneity in bacterial populations.

The MazF toxin sequence-specifically cleaves single-stranded RNA upon various stressful conditions, and it is activated as a part of the mazEF toxin-antitoxin module in Escherichia coli. Although autoregulation of mazEF expression through the MazE antitoxin-dependent transcriptional repression has been biochemically characterized, less is known about post-transcriptional autoregulation, as well as how both of these autoregulatory features affect growth of single cells during conditions that promote MazF production. Here, we demonstrate post-transcriptional autoregulation of mazF expression dynamics by MazF cleaving its own transcript. Single-cell analyses of bacterial populations during ectopic MazF production indicated that two-level autoregulation of mazEF expression influences cell-to-cell growth rate heterogeneity. The increase in growth rate heterogeneity is governed by the MazE antitoxin, and tuned by the MazF-dependent mazF mRNA cleavage. Also, both autoregulatory features grant rapid exit from the stress caused by mazF overexpression. Time-lapse microscopy revealed that MazF-mediated cleavage of mazF mRNA leads to increased temporal variability in length of individual cells during ectopic mazF overexpression, as explained by a stochastic model indicating that mazEF mRNA cleavage underlies temporal fluctuations in MazF levels during stress.

The RNA ligase RtcB reverses MazF-induced ribosome heterogeneity in Escherichia coli.

When Escherichia coli encounters stress, the endoribonuclease MazF initiates a post-transcriptional response that results in the reprogramming of protein synthesis. By removing the 3΄-terminus of the 16S rRNA, MazF generates specialized ribosomes that selectively translate mRNAs likewise processed by MazF. Given the energy required for de novo ribosome biosynthesis, we considered the existence of a repair mechanism operating upon stress relief to recycle the modified ribosomes. Here, we show that the stress-ribosomes and the 3΄-terminal 16S rRNA fragment are stable during adverse conditions. Moreover, employing in vitro and in vivo approaches we demonstrate that the RNA ligase RtcB catalyzes the re-ligation of the truncated 16S rRNA present in specialized ribosomes Thereby their ability to translate canonical mRNAs is fully restored. Together, our findings not only provide a physiological function for the RNA ligase RtcB in bacteria but highlight the reversibility of ribosome heterogeneity, a crucial but hitherto undescribed concept for translational regulation.

The MazF-regulon: a toolbox for the post-transcriptional stress response in Escherichia coli.

Flexible adaptation to environmental stress is vital for bacteria. An energy-efficient post-transcriptional stress response mechanism in Escherichia coli is governed by the toxin MazF. After stress-induced activation the endoribonuclease MazF processes a distinct subset of transcripts as well as the 16S ribosomal RNA in the context of mature ribosomes. As these 'stress-ribosomes' are specific for the MazF-processed mRNAs, the translational program is changed. To identify this 'MazF-regulon' we employed Poly-seq (polysome fractionation coupled with RNA-seq analysis) and analyzed alterations introduced into the transcriptome and translatome after mazF overexpression. Unexpectedly, our results reveal that the corresponding protein products are involved in all cellular processes and do not particularly contribute to the general stress response. Moreover, our findings suggest that translational reprogramming serves as a fast-track reaction to harsh stress and highlight the so far underestimated significance of selective translation as a global regulatory mechanism in gene expression. Considering the reported implication of toxin-antitoxin (TA) systems in persistence, our results indicate that MazF acts as a prime effector during harsh stress that potentially introduces translational heterogeneity within a bacterial population thereby stimulating persister cell formation.

An unexpected type of ribosomes induced by kasugamycin: a look into ancestral times of protein synthesis?

Translation of leaderless mRNAs, lacking ribosomal recruitment signals other than the 5'-terminal AUG-initiating codon, occurs in all three domains of life. Contemporary leaderless mRNAs may therefore be viewed as molecular fossils resembling ancestral mRNAs. Here, we analyzed the phenomenon of sustained translation of a leaderless mRNA in the presence of the antibiotic kasugamycin. Unexpected from the known in vitro effects of the drug, kasugamycin induced the formation of stable approximately 61S ribosomes in vivo, which were proficient in selectively translating leaderless mRNA. 61S particles are devoid of more than six proteins of the small subunit, including the functionally important proteins S1 and S12. The lack of these proteins could be reconciled with structural changes in the 16S rRNA. These studies provide in vivo evidence for the functionality of ribosomes devoid of multiple proteins and shed light on the evolutionary history of ribosomes.

Structural basis for the interaction of protein S1 with the Escherichia coli ribosome.

In Gram-negative bacteria, the multi-domain protein S1 is essential for translation initiation, as it recruits the mRNA and facilitates its localization in the decoding centre. In sharp contrast to its functional importance, S1 is still lacking from the high-resolution structures available for Escherichia coli and Thermus thermophilus ribosomes and thus the molecular mechanism governing the S1-ribosome interaction has still remained elusive. Here, we present the structure of the N-terminal S1 domain D1 when bound to the ribosome at atomic resolution by using a combination of NMR, X-ray crystallography and cryo-electron microscopy. Together with biochemical assays, the structure reveals that S1 is anchored to the ribosome primarily via a stabilizing π-stacking interaction within the short but conserved N-terminal segment that is flexibly connected to domain D1. This interaction is further stabilized by salt bridges involving the zinc binding pocket of protein S2. Overall, this work provides one hitherto enigmatic piece in the 'ribosome puzzle', namely the detailed molecular insight into the topology of the S1-ribosome interface. Moreover, our data suggest novel mechanisms that have the potential to modulate protein synthesis in response to environmental cues by changing the affinity of S1 for the ribosome.

Selective translation during stress in Escherichia coli.

The bacterial stress response, a strategy to cope with environmental changes, is generally known to operate on the transcriptional level. Here, we discuss a novel paradigm for stress adaptation at the post-transcriptional level, based on the recent discovery of a stress-induced modified form of the translation machinery in Escherichia coli that is generated by MazF, the toxin component of the toxin-antitoxin (TA) module mazEF. Under stress, the induced endoribonuclease MazF removes the 3'-terminal 43 nucleotides of the 16S rRNA of ribosomes and, concomitantly, the 5'-untranslated regions (UTRs) of specific transcripts. This elegant mechanism enables selective translation due to the complementary effect of MazF on ribosomes and mRNAs, and also represents the first example of functional ribosome heterogeneity based on rRNA alteration.

Catabolite repression control protein antagonist, a novel player in Pseudomonas aeruginosa carbon catabolite repression control.

In the opportunistic human pathogen Pseudomonas aeruginosa (Pae), carbon catabolite repression (CCR) orchestrates the hierarchical utilization of N and C sources, and impacts virulence, antibiotic resistance and biofilm development. During CCR, the RNA chaperone Hfq and the catabolite repression control protein Crc form assemblies on target mRNAs that impede translation of proteins involved in uptake and catabolism of less preferred C sources. After exhaustion of the preferred C-source, translational repression of target genes is relieved by the regulatory RNA CrcZ, which binds to and acts as a decoy for Hfq. Here, we asked whether Crc action can be modulated to relieve CCR after exhaustion of a preferred carbon source. As Crc does not bind to RNA per se, we endeavored to identify an interacting protein. In vivo co-purification studies, co-immunoprecipitation and biophysical assays revealed that Crc binds to Pae strain O1 protein PA1677. Our structural studies support bioinformatics analyzes showing that PA1677 belongs to the isochorismatase-like superfamily. Ectopic expression of PA1677 resulted in de-repression of Hfq/Crc controlled target genes, while in the absence of the protein, an extended lag phase is observed during diauxic growth on a preferred and a non-preferred carbon source. This observations indicate that PA1677 acts as an antagonist of Crc that favors synthesis of proteins required to metabolize non-preferred carbon sources. We present a working model wherein PA1677 diminishes the formation of productive Hfq/Crc repressive complexes on target mRNAs by titrating Crc. Accordingly, we propose the name CrcA (catabolite repression control protein antagonist) for PA1677.

Quantifying heterologous gene expression during ectopic MazF production in Escherichia coli.

OBJECTIVE: MazF is a sequence-specific endoribonuclease-toxin of the MazEF toxin-antitoxin system. MazF cleaves single-stranded ribonucleic acid (RNA) regions at adenine-cytosine-adenine (ACA) sequences in the bacterium Escherichia coli. The MazEF system has been used in various biotechnology and synthetic biology applications. In this study, we infer how ectopic mazF overexpression affects production of heterologous proteins. To this end, we quantified the levels of fluorescent proteins expressed in E. coli from reporters translated from the ACA-containing or ACA-less messenger RNAs (mRNAs). Additionally, we addressed the impact of the 5'-untranslated region of these reporter mRNAs under the same conditions by comparing expression from mRNAs that comprise (canonical mRNA) or lack this region (leaderless mRNA). RESULTS: Flow cytometry analysis indicates that during mazF overexpression, fluorescent proteins are translated from the canonical as well as leaderless mRNAs. Our analysis further indicates that longer mazF overexpression generally increases the concentration of fluorescent proteins translated from ACA-less mRNAs, however it also substantially increases bacterial population heterogeneity. Finally, our results suggest that the strength and duration of mazF overexpression should be optimized for each experimental setup, to maximize the heterologous protein production and minimize the amount of phenotypic heterogeneity in bacterial populations, which is unfavorable in biotechnological processes.

In vitro analysis of the interaction between the small RNA SR1 and its primary target ahrC mRNA.

Small regulatory RNAs (sRNAs) from bacterial chromosomes became the focus of research over the past five years. However, relatively little is known in terms of structural requirements, kinetics of interaction with their targets and degradation in contrast to well-studied plasmid-encoded antisense RNAs. Here, we present a detailed in vitro analysis of SR1, a sRNA of Bacillus subtilis that is involved in regulation of arginine catabolism by basepairing with its target, ahrC mRNA. The secondary structures of SR1 species of different lengths and of the SR1/ahrC RNA complex were determined and functional segments required for complex formation narrowed down. The initial contact between SR1 and its target was shown to involve the 5' part of the SR1 terminator stem and a region 100 bp downstream from the ahrC transcriptional start site. Toeprinting studies and secondary structure probing of the ahrC/SR1 complex indicated that SR1 inhibits translation initiation by inducing structural changes downstream from the ahrC RBS. Furthermore, it was demonstrated that Hfq, which binds both SR1 and ahrC RNA was not required to promote ahrC/SR1 complex formation but to enable the translation of ahrC mRNA. The intracellular concentrations of SR1 were calculated under different growth conditions.

Leaderless mRNAs in the Spotlight: Ancient but Not Outdated!

Previously, leaderless mRNAs (lmRNAs) were perceived to make up only a minor fraction of the transcriptome in bacteria. However, advancements in RNA sequencing technology are uncovering vast numbers of lmRNAs, particularly in archaea, Actinobacteria, and extremophiles and thus underline their significance in cellular physiology and regulation. Due to the absence of conventional ribosome binding signals, lmRNA translation initiation is distinct from canonical mRNAs and can therefore be differentially regulated. The ribosome's inherent ability to bind a 5'-terminal AUG can stabilize and protect the lmRNA from degradation or allow ribosomal loading for downstream initiation events. As a result, lmRNAs remain translationally competent during a variety of physiological conditions, allowing them to contribute to multiple regulatory mechanisms. Furthermore, the abundance of lmRNAs can increase during adverse conditions through the upregulation of lmRNA transcription from alternative promoters or by the generation of lmRNAs from canonical mRNAs cleaved by an endonucleolytic toxin. In these ways, lmRNA translation can continue during stress and contribute to regulation, illustrating their importance in the cell. Due to their presence in all domains of life and their ability to be translated by heterologous hosts, lmRNAs appear further to represent ancestral transcripts that might allow us to study the evolution of the ribosome and the translational process.

Coordinated Control of mRNA and rRNA Processing Controls Embryonic Stem Cell Pluripotency and Differentiation.

Stem cell-specific transcriptional networks are well known to control pluripotency, but constitutive cellular processes such as mRNA splicing and protein synthesis can add complex layers of regulation with poorly understood effects on cell-fate decisions. Here, we show that the RNA binding protein HTATSF1 controls embryonic stem cell differentiation by regulating multiple aspects of RNA processing during ribosome biogenesis. HTATSF1, in a complex with splicing factor SF3B1, controls intron removal from ribosomal protein transcripts and regulates ribosomal RNA transcription and processing, thereby controlling 60S ribosomal abundance and protein synthesis. HTATSF1-dependent protein synthesis is essential for naive pre-implantation epiblast to transition into post-implantation epiblast, a stage with transiently low protein synthesis, and further differentiation toward neuroectoderm. Together, these results identify coordinated regulation of ribosomal RNA and protein synthesis by HTATSF1 and show that this essential mechanism controls protein synthesis during early mammalian embryogenesis.

Both RNase E and RNase III control the stability of sodB mRNA upon translational inhibition by the small regulatory RNA RyhB.

Previous work has demonstrated that iron-dependent variations in the steady-state concentration and translatability of sodB mRNA are modulated by the small regulatory RNA RyhB, the RNA chaperone Hfq and RNase E. In agreement with the proposed role of RNase E, we found that the decay of sodB mRNA is retarded upon inactivation of RNase E in vivo, and that the enzyme cleaves within the sodB 5'-untranslated region (5'-UTR) in vitro, thereby removing the 5' stem-loop structure that facilitates Hfq and ribosome binding. Moreover, RNase E cleavage can also occur at a cryptic site that becomes available upon sodB 5'-UTR/RyhB base pairing. We show that while playing an important role in facilitating the interaction of RyhB with sodB mRNA, Hfq is not tightly retained by the RyhB-sodB mRNA complex and can be released from it through interaction with other RNAs added in trans. Unlike turnover of sodB mRNA, RyhB decay in vivo is mainly dependent on RNase III, and its cleavage by RNase III in vitro is facilitated upon base pairing with the sodB 5'-UTR. These data are discussed in terms of a model, which accounts for the observed roles of RNase E and RNase III in sodB mRNA turnover.

MazF activation promotes translational heterogeneity of the grcA mRNA in Escherichia coli populations.

Bacteria adapt to adverse environmental conditions by altering gene expression patterns. Recently, a novel stress adaptation mechanism has been described that allows Escherichia coli to alter gene expression at the post-transcriptional level. The key player in this regulatory pathway is the endoribonuclease MazF, the toxin component of the toxin-antitoxin module mazEF that is triggered by various stressful conditions. In general, MazF degrades the majority of transcripts by cleaving at ACA sites, which results in the retardation of bacterial growth. Furthermore, MazF can process a small subset of mRNAs and render them leaderless by removing their ribosome binding site. MazF concomitantly modifies ribosomes, making them selective for the translation of leaderless mRNAs. In this study, we employed fluorescent reporter-systems to investigate mazEF expression during stressful conditions, and to infer consequences of the mRNA processing mediated by MazF on gene expression at the single-cell level. Our results suggest that mazEF transcription is maintained at low levels in single cells encountering adverse conditions, such as antibiotic stress or amino acid starvation. Moreover, using the grcA mRNA as a model for MazF-mediated mRNA processing, we found that MazF activation promotes heterogeneity in the grcA reporter expression, resulting in a subpopulation of cells with increased levels of GrcA reporter protein.

Translation initiation with 70S ribosomes: an alternative pathway for leaderless mRNAs.

It is generally accepted that translation in bacteria is initiated by 30S ribosomal subunits. In contrast, several lines of rather indirect in vitro evidence suggest that 70S monosomes are capable of initiating translation of leaderless mRNAs, starting with the A of the initiation codon. In this study, we demonstrate the proficiency of dedicated 70S ribosomes in in vitro translation of leaderless mRNAs. In support, we show that a natural leaderless mRNA can be translated with crosslinked 70S wild-type ribosomes. Moreover, we report that leaderless mRNA translation continues under conditions where the prevalence of 70S ribosomes is created in vivo, and where translation of bulk mRNA ceases. These studies provide in vivo as well as direct in vitro evidence for a 70S initiation pathway of a naturally occurring leaderless mRNA, and are discussed in light of their significance for bacterial growth under adverse conditions and their evolutionary implications for translation.

Insights into the Stress Response Triggered by Kasugamycin in Escherichia coli.

The bacteriostatic aminoglycoside antibiotic kasugamycin inhibits protein synthesis at an initial step without affecting translation elongation. It binds to the mRNA track of the ribosome and prevents formation of the translation initiation complex on canonical mRNAs. In contrast, translation of leaderless mRNAs continues in the presence of the drug in vivo. Previously, we have shown that kasugamycin treatment in E. coli stimulates the formation of protein-depleted ribosomes that are selective for leaderless mRNAs. Here, we provide evidence that prolonged kasugamycin treatment leads to selective synthesis of specific proteins. Our studies indicate that leaderless and short-leadered mRNAs are generated by different molecular mechanisms including alternative transcription and RNA processing. Moreover, we provide evidence for ribosome heterogeneity in response to kasugamycin treatment by alteration of the modification status of the stalk proteins bL7/L12.

A Stress-Induced Bias in the Reading of the Genetic Code in Escherichia coli.

Escherichia coli mazEF is an extensively studied stress-induced toxin-antitoxin (TA) system. The toxin MazF is an endoribonuclease that cleaves RNAs at ACA sites. Thereby, under stress, the induced MazF generates a stress-induced translation machinery (STM), composed of MazF-processed mRNAs and selective ribosomes that specifically translate the processed mRNAs. Here, we further characterized the STM system, finding that MazF cleaves only ACA sites located in the open reading frames of processed mRNAs, while out-of-frame ACAs are resistant. This in-frame ACA cleavage of MazF seems to depend on MazF binding to an extracellular-death-factor (EDF)-like element in ribosomal protein bS1 (bacterial S1), apparently causing MazF to be part of STM ribosomes. Furthermore, due to the in-frame MazF cleavage of ACAs under stress, a bias occurs in the reading of the genetic code causing the amino acid threonine to be encoded only by its synonym codon ACC, ACU, or ACG, instead of by ACA. IMPORTANCE: The genetic code is a universal characteristic of all living organisms. It defines the set of rules by which nucleotide triplets specify which amino acid will be incorporated into a protein. Our results represent the first existing report on a stress-induced bias in the reading of the genetic code. We found that in E. coli, under stress, the amino acid threonine is encoded only by its synonym codon ACC, ACU, or ACG, instead of by ACA. This is because under stress, MazF generates a stress-induced translation machinery (STM) in which MazF cleaves in-frame ACA sites of the processed mRNAs.

Escherichia coli Quorum-Sensing EDF, A Peptide Generated by Novel Multiple Distinct Mechanisms and Regulated by trans-Translation.

UNLABELLED: Eshcerichia coli mazEF is a stress-induced toxin-antitoxin module mediating cell death and requiring a quorum-sensing (QS) extracellular death factor (EDF), the pentapeptide NNWNN. Here we uncovered several distinct molecular mechanisms involved in its generation from the zwf mRNA encoding glucose-6-phosphate dehydrogenase. In particular, we show that, under stress conditions, the endoribonuclease MazF cleaves specific ACA sites, thereby generating a leaderless zwf mRNA which is truncated 30 codons after the EDF-encoding region. Since the nascent ribosome peptide exit tunnel can accommodate up to 40 amino acids, this arrangement allows the localization of the EDF residues inside the tunnel when the ribosome is stalled at the truncation site. Moreover, ribosome stalling activates the trans-translation system, which provides a means for the involvement of ClpPX in EDF generation. Furthermore, the trans-translation is described as a regulatory system that attenuated the generation of EDF, leading to low levels of EDF in the single cell. Therefore, the threshold EDF molecule concentration required is achieved only by the whole population, as expected for QS. IMPORTANCE: Bacteria communicate with one another via quorum-sensing (QS) signal molecules. QS provides a mechanism for bacteria to monitor each other's presence and to modulate gene expression in response to population density. Previously, we added E. coli pentapeptide EDF to this list of QS molecules. We showed that, under stress conditions, the induced MazF, an endoribonuclease cleaving at ACA sites, generates EDF from zwf. Here we studied the mechanism of EDF generation and asked whether it is related to EDF density dependency. We illustrated that, under stress conditions, multiple distinct complex mechanisms are involved in EDF generation. This includes formation of leaderless truncated zwf mRNA by MazF, configuration of a length corresponding to the nascent ribosome peptide exit tunnel, rescue performed by the trans-translation system, and cleavage by ClpPX protease. trans-Translation is described as a regulatory system attenuating EDF generation and leading to low levels of EDF in the single cell, as expected for QS.