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Selective translation by alternative bacterial ribosomes.

ABSTRACT: Alternative ribosome subunit proteins are prevalent in the genomes of diverse bacterial species, but their functional significance is controversial. Attempts to study microbial ribosomal heterogeneity have mostly relied on comparing wild-type strains with mutants in which subunits have been deleted, but this approach does not allow direct comparison of alternate ribosome isoforms isolated from identical cellular contexts. Here, by simultaneously purifying canonical and alternative RpsR ribosomes from Mycobacterium smegmatis, we show that alternative ribosomes have distinct translational features compared with their canonical counterparts. Both alternative and canonical ribosomes actively take part in protein synthesis, although they translate a subset of genes with differential efficiency as measured by ribosome profiling. We also show that alternative ribosomes have a relative defect in initiation complex formation. Furthermore, a strain of M. smegmatis in which the alternative ribosome protein operon is deleted grows poorly in iron-depleted medium, uncovering a role for alternative ribosomes in iron homeostasis. Our work confirms the distinct and nonredundant contribution of alternative bacterial ribosomes for adaptation to hostile environments.

SUBMITTER: Chen YX

PROVIDER: S-EPMC7431078 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Selective translation by alternative bacterial ribosomes

Project description:Mycobacteria encode several additional alternative ribosomal proteins (AltRP) and the funtion of AltRP invovling in translation is little known. In this study, we investigate the different translation landscape of AltRibo and CanRibo and found AltRibo have a relative 5' reads accumlation and preferential codon usage.

2019-04-11 | GSE127827 | GEO

Selective translation by alternative bacterial ribosomes

Project description:Selective translation by alternative bacterial ribosomes

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Alternative fates of paused ribosomes during translation termination.

Project description:The bacterial tmRNA·SmpB system facilitates recycling of stalled translational complexes in a process termed "ribosome rescue." During ribosome rescue, the nascent chain is tagged with the tmRNA-encoded ssrA peptide, which targets the tagged polypeptide for degradation. Translational pausing also induces a variety of recoding events such as frameshifts, ribosome hops, and stop codon readthrough. To examine the interplay between recoding and ribosome rescue, we determined the various fates of ribosomes that pause during translation termination. We expressed a model protein containing the C-terminal Asp-Pro nascent peptide motif (which interferes with translation termination) and quantified the protein chains produced by recoding and ssrA-peptide tagging. The nature and extent of translational recoding depended upon the codon for the C-terminal Pro residue, with CCU and CCC promoting efficient +1 frameshifting. In contrast, ssrA-peptide tagging was unaffected by C-terminal Pro coding. Moreover, +1 frameshifting was not suppressed by tmRNA·SmpB activity, suggesting that recoding and ribosome rescue are not competing events. However, cells lacking ribosomal protein L9 (?L9) exhibited a significant increase in recoding and a concomitant decrease in ssrA-peptide tagging. Pulse-chase analysis revealed that pre-termination ribosomes turn over more rapidly in ?L9 cells, suggesting that increased recoding alleviates the translational arrest. Together, these results indicate that tmRNA·SmpB does not suppress transient ribosome pauses, but responds to prolonged translational arrest.

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Specialized yeast ribosomes: a customized tool for selective mRNA translation.

Project description:Evidence is now accumulating that sub-populations of ribosomes - so-called specialized ribosomes - can favour the translation of subsets of mRNAs. Here we use a large collection of diploid yeast strains, each deficient in one or other copy of the set of ribosomal protein (RP) genes, to generate eukaryotic cells carrying distinct populations of altered 'specialized' ribosomes. We show by comparative protein synthesis assays that different heterologous mRNA reporters based on luciferase are preferentially translated by distinct populations of specialized ribosomes. These mRNAs include reporters carrying premature termination codons (PTC) thus allowing us to identify specialized ribosomes that alter the efficiency of translation termination leading to enhanced synthesis of the wild-type protein. This finding suggests that these strains can be used to identify novel therapeutic targets in the ribosome. To explore this further we examined the translation of the mRNA encoding the extracellular matrix protein laminin ?3 (LAMB3) since a LAMB3-PTC mutant is implicated in the blistering skin disease Epidermolysis bullosa (EB). This screen identified specialized ribosomes with reduced levels of RP L35B as showing enhanced synthesis of full-length LAMB3 in cells expressing the LAMB3-PTC mutant. Importantly, the RP L35B sub-population of specialized ribosomes leave both translation of a reporter luciferase carrying a different PTC and bulk mRNA translation largely unaltered.

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Binding of 2',3'-Cyclic Nucleotide Monophosphates to Bacterial Ribosomes Inhibits Translation.

Project description:The intracellular small molecules 2',3'-cyclic nucleotide monophosphates (2',3'-cNMPs) have recently been rediscovered within both prokaryotes and eukaryotes. Studies in bacteria have demonstrated that 2',3'-cNMP levels affect bacterial phenotypes, such as biofilm formation, motility, and growth, and modulate expression of numerous genes, suggesting that 2',3'-cNMP levels are monitored by cells. In this study, 2',3'-cNMP-linked affinity chromatography resins were used to identify Escherichia coli proteins that bind 2',3'-cNMPs, with the top hits including all of the ribosomal proteins, and to confirm direct binding of purified ribosomes. Using in vitro translation assays, we have demonstrated that 2',3'-cNMPs inhibit translation at concentrations found in amino acid-starved cells. In addition, a genetically encoded tool to increase cellular 2',3'-cNMP levels was developed and was demonstrated to decrease E. coli growth rates. Taken together, this work suggests a mechanism for 2',3-cNMP levels to modulate bacterial phenotypes by rapidly affecting translation.

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

Project description: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.

| S-EPMC4894548 | biostudies-literature

Relaxed initiation pausing of ribosomes drives oncogenic translation.

Project description:Translation is a crucial process in cancer development and progression. Many oncogenic signaling pathways target the translation initiation stage to satisfy the increased anabolic demands of cancer cells. Using quantitative profiling of initiating ribosomes, we found that ribosomal pausing at the start codon serves as a "brake" to restrain the translational output. In response to oncogenic RAS signaling, the initiation pausing relaxes and contributes to the increased translational flux. Intriguingly, messenger RNA (mRNA) m6A modification in the vicinity of start codons influences the behavior of initiating ribosomes. Under oncogenic RAS signaling, the reduced mRNA methylation leads to relaxed initiation pausing, thereby promoting malignant transformation and tumor growth. Restored initiation pausing by inhibiting m6A demethylases suppresses RAS-mediated oncogenic translation and subsequent tumorigenesis. Our findings unveil a paradigm of translational control that is co-opted by RAS mutant cancer cells to drive malignant phenotypes.

| S-EPMC7888950 | biostudies-literature

RACK1 Specifically Regulates Translation through Its Binding to Ribosomes.

Project description:The translational capability of ribosomes deprived of specific nonfundamental ribosomal proteins may be altered. Physiological mechanisms are scanty, and it is unclear whether free ribosomal proteins can cross talk with the signaling machinery. RACK1 (receptor for activated C kinase 1) is a highly conserved scaffold protein, located on the 40S subunit near the mRNA exit channel. RACK1 is involved in a variety of intracellular contexts, both on and off the ribosomes, acting as a receptor for proteins in signaling, such as the protein kinase C (PKC) family. Here we show that the binding of RACK1 to ribosomes is essential for full translation of capped mRNAs and efficient recruitment of eukaryotic initiation factor 4E (eIF4E). In vitro, when RACK1 is partially depleted, supplementing the ribosome machinery with wild-type RACK1 restores the translational capability, whereas the addition of a RACK1 mutant that is unable to bind ribosomes does not. Outside the ribosome, RACK1 has a reduced half-life. By accumulating in living cells, free RACK1 exerts an inhibitory phenotype, impairing cell cycle progression and repressing global translation. Here we present RACK1 binding to ribosomes as a crucial way to regulate translation, possibly through interaction with known partners on or off the ribosome that are involved in signaling.

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Selective recruitment of stress-responsive mRNAs to ribosomes for translation by acetylated protein S1 during nutrient stress in Escherichia coli.

Project description:The chemical modification of ribosomes plays an important regulatory role in cellular translation adaptation in response to environmental stresses. Nevertheless, how the modified ribosome reprograms the translation machinery for the preferential expression of the specific mRNAs encoding stress-responsive proteins to stress remains poorly understood. Here, we find that AcP-induced acetylation of K411 and K464 in ribosomal protein S1 during carbon-nitrogen imbalance, which in turn impacts its binding with distinct mRNAs. S1 acetylation shows differential selectivity for recruiting subsets of mRNAs to ribosomes. Using the RNC-Seq method, we find that mimic acetylated S1 prefers transcripts related with the formation of flagella/biofilms, two-component systems, nitrogen assimilation, amino acid degradation, and lipopolysaccharide biosynthesis, whereas inhibits the translation of mRNAs involved in amino acid biosynthesis and most ribosomal proteins. Importantly, further characterization of S1-binding site (SBS) sequences of mRNAs with different translation efficiencies indicated that the presence of a conserved motif allows coordinated regulation of S1 acetylation-driven translation reprogramming for cell survival during nitrogen starvation. These findings expand the repertoire of ribosome heterogeneity to the acetylation level of S1 at specific sites and its role in the ribosome-mediated regulation of gene expression as a cellular response at the translational level to stress.

| S-EPMC9437053 | biostudies-literature

Bacterial toxin HigB associates with ribosomes and mediates translation-dependent mRNA cleavage at A-rich sites.

Project description:Most pathogenic Proteus species are primarily associated with urinary tract infections, especially in persons with indwelling catheters or functional/anatomic abnormalities of the urinary tract. Urinary tract infections caused by Proteus vulgaris typically form biofilms and are resistant to commonly used antibiotics. The Rts1 conjugative plasmid from a clinical isolate of P. vulgaris carries over 300 predicted open reading frames, including antibiotic resistance genes. The maintenance of the Rts1 plasmid is ensured in part by the HigBA toxin-antitoxin system. We determined the precise mechanism of action of the HigB toxin in vivo, which is distinct from other known toxins. We demonstrate that HigB is an endoribonuclease whose enzymatic activity is dependent on association with ribosomes through the 50 S subunit. Using primer extension analysis of several test mRNAs, we showed that HigB cleaved extensively across the entire length of coding regions only at specific recognition sequences. HigB mediated cleavage of 100% of both in-frame and out-of-frame AAA sequences. In addition, HigB cleaved approximately 20% of AA sequences in coding regions and occasionally cut single As. Remarkably, the cleavage specificity of HigB coincided with one of the most frequently used codons in the AT-rich Proteus spp., AAA (lysine). Therefore, the HigB-mediated plasmid maintenance system for the Rts1 plasmid highlights the intimate relationship between host cells and extrachromosomal DNA that enables the dynamic acquisition of genes that impart a spectrum of survival advantages, including those encoding multidrug resistance and virulence factors.

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