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Mammalian mitochondria have their own dedicated protein synthesis system, which produces 13 essential subunits of the oxidative phosphorylation complexes. Here, we describe the in vitro reconstitution of the mammalian mitochondrial translation system, utilizing purified recombinant mitochondrial translation factors, 55S ribosomes from pig liver mitochondria, and a heterologous yeast tRNA mixture. The system is capable of translating leaderless mRNAs encoding model proteins, such as nanoluciferase with a molecular weight of 19 kDa, and is readily applicable for in vitro evaluations of mRNAs and nascent peptide chain sequences, as well as factors and small molecules that affect mitochondrial translation.

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The genus Mycobacterium contains several slow-growing human pathogens, including Mycobacterium tuberculosis, Mycobacterium leprae, and Mycobacterium avium. Mycobacterium smegmatis is a nonpathogenic and fast growing species within this genus. In 1990, a mutant of M. smegmatis, designated mc2155, that could be transformed with episomal plasmids was isolated, elevating M. smegmatis to model status as the ideal surrogate for mycobacterial research. Classical bacterial models, such as Escherichia coli, were inadequate for mycobacteria research because they have low genetic conservation, different physiology, and lack the novel envelope structure that distinguishes the Mycobacterium genus. By contrast, M. smegmatis encodes thousands of conserved mycobacterial gene orthologs and has the same cell architecture and physiology. Dissection and characterization of conserved genes, structures, and processes in genetically tractable M. smegmatis mc2155 have since provided previously unattainable insights on these same features in its slow-growing relatives. Notably, tuberculosis (TB) drugs, including the first-line drugs isoniazid and ethambutol, are active against M. smegmatis, but not against E. coli, allowing the identification of their physiological targets. Furthermore, Bedaquiline, the first new TB drug in 40 years, was discovered through an M. smegmatis screen. M. smegmatis has become a model bacterium, not only for M. tuberculosis, but for all other Mycobacterium species and related genera. With a repertoire of bioinformatic and physical resources, including the recently established Mycobacterial Systems Resource, M. smegmatis will continue to accelerate mycobacterial research and advance the field of microbiology.

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A strategy that can be applied to the research of new molecules with antibacterial activity is to look for inhibitors of essential bacterial processes within large collections of chemically heterogeneous compounds. The implementation of this approach requires the development of assays aimed at the identification of molecules interfering with specific cell pathways that can also be used in high-throughput analysis of large chemical libraries. Here, we describe a fluorescence-based whole-cell assay in Escherichia coli devised to find inhibitors of the translation initiation pathway. Translation is a complex and essential mechanism. It involves numerous sub-steps performed by factors that are in many cases sufficiently dissimilar in bacterial and eukaryotic cells to be targetable with domain-specific drugs. As a matter of fact, translation has been proven as one of the few bacterial mechanisms pharmacologically tractable with specific antibiotics. The assay described in this updated chapter is tailored to the identification of molecules affecting the first stage of translation initiation, which is the most dissimilar step in bacteria versus mammals. The effect of the compounds under analysis is measured in living cells, thus allowing evaluation of their in vivo performance as inhibitors of translation initiation. Compared with other assays for antibacterials, the major advantages of this screen are its simplicity, high mechanism specificity, and amenability to scaling up to high-throughput analyses.

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In bacteria, the translation of genetic information can begin through at least three different mechanisms: canonical or Shine-Dalgarno-led initiation, readthrough or 70S scanning initiation, or leaderless initiation. Here, we discuss the main features and regulation of the last, which is characterized mainly by the ability of 70S ribosomal particles to bind to AUG located at or near the 5' end of mRNAs to initiate translation. These leaderless mRNAs (lmRNAs) are rare in enterobacteria, such as Escherichia coli, but are common in other bacteria, such as Mycobacterium tuberculosis and Deinococcus deserti, where they may represent more than 20% and even up to 60% of the genes. Given that lmRNAs are devoid of a 5' untranslated region and the Shine-Dalgarno sequence located within it, the mechanism of translation regulation must depend on molecular strategies that are different from what has been observed in the Shine-Dalgarno-led translation. Diverse regulatory mechanisms have been proposed, including the processing of ribosomal RNA and changes in the abundance of translation factors, but all of them produce global changes in the initiation of lmRNA translation. Thus, further research will be required to understand how the initiation of the translation of particular lmRNA genes is regulated.

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A tRNA-like sequence conserved in the genomes of all Escherichia coli strains was found. The sequence resembles arginine-tRNA, which is present in E. coli pathogenic islands and phages. Expression experiments revealed that this sequence is a part of a leaderless mRNA encoding a short peptide (60 amino acids: XtpA). A deletion mutant of this gene is more sensitive than wild-type cell to several aminoglycoside antibiotics at low concentrations. Further analyses indicated that XtpA positively regulates the expression of GcvB small RNA, which is involved in the intrinsic resistance to aminoblycosides in E. coli.

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Mycobacterium tuberculosis (Mtb) causes one of humankind's deadliest diseases, tuberculosis. Mtb protein synthesis machinery possesses several unique species-specific features, including its ribosome that carries two mycobacterial specific ribosomal proteins, bL37 and bS22, and ribosomal RNA segments. Since the protein synthesis is a vital cellular process that occurs on the ribosome, a detailed knowledge of the structure and function of mycobacterial ribosomes is essential to understand the cell's proteome by translation regulation. Like in many bacterial species such as Bacillus subtilis and Streptomyces coelicolor, two distinct populations of ribosomes have been identified in Mtb. Under low-zinc conditions, Mtb ribosomal proteins S14, S18, L28, and L33 are replaced with their non-zinc binding paralogues. Depending upon the nature of physiological stress, species-specific modulation of translation by stress factors and toxins that interact with the ribosome have been reported. In addition, about one-fourth of messenger RNAs in mycobacteria have been reported to be leaderless, i.e., without 5' UTR regions. However, the mechanism by which they are recruited to the Mtb ribosome is not understood. In this review, we highlight the mycobacteria-specific features of the translation apparatus and propose exploiting these features to improve the efficacy and specificity of existing antibiotics used to treat tuberculosis.

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The universally conserved P-loop GTPases control diverse cellular processes, like signal transduction, ribosome assembly, cell motility, and intracellular transport and translation. YchF belongs to the Obg-family of P-loop GTPases and is one of the least characterized member of this family. It is unique because it preferentially hydrolyses ATP rather than GTP, but its physiological role is largely unknown. Studies in different organisms including humans suggest a possible role of YchF in regulating the cellular adaptation to stress conditions. In the current study, we explored the role of YchF in the model organism Escherichia coli. By western blot and promoter fusion experiments, we demonstrate that YchF levels decrease during stress conditions or when cells enter stationary phase. The decline in YchF levels trigger increased stress resistance and cells lacking YchF are resistant to multiple stress conditions, like oxidative stress, replication stress, or translational stress. By in vivo site directed cross-linking we demonstrate that YchF interacts with the translation initiation factor 3 (IF3) and with multiple ribosomal proteins at the surface of the small ribosomal subunit. The absence of YchF enhances the anti-association activity of IF3, stimulates the translation of leaderless mRNAs, and increases the resistance against the endoribonuclease MazF, which generates leaderless mRNAs during stress conditions. In summary, our data identify YchF as a stress-responsive regulator of leaderless mRNA translation.

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The ability to respond to metabolic or environmental changes is an essential feature in all cells and involves both transcriptional and translational regulators that adjust the metabolic activity to fluctuating conditions. While transcriptional regulation has been studied in detail, the important role of the ribosome as an additional player in regulating gene expression is only beginning to emerge. Ribosome-interacting proteins are central to this translational regulation and include universally conserved ribosome interacting proteins, such as the ATPase YchF (Ola1 in eukaryotes). In both eukaryotes and bacteria, the cellular concentrations of YchF/Ola1 determine the ability to cope with different stress conditions and are linked to several pathologies in humans. The available data indicate that YchF/Ola1 regulates the stress response via controlling non-canonical translation initiation and via protein degradation. Although the molecular mechanisms appear to be different between bacteria and eukaryotes, increased non-canonical translation initiation is a common consequence of YchF/Ola1 regulated translational control in E. coli and H. sapiens. In this review, we summarize recent insights into the role of the universally conserved ATPase YchF/Ola1 in adapting translation to unfavourable conditions.

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The mitochondrial genome of Chlamydomonas reinhardtii encodes eight protein coding genes transcribed on two polycistronic primary transcripts. The mRNAs are endonucleolytically cleaved from these transcripts directly upstream of their AUG start codons, creating leaderless mRNAs with 3' untranslated regions (UTR) comprised of most or all of their downstream intergenic regions. In this report, we provide evidence that these processed linear mRNAs are circularized, which places the 3' UTR upstream of the 5' start codon, creating a leader sequence ex post facto. The circular mRNAs were found to be ribosome associate by polysome profiling experiments suggesting they are translated. Sequencing of the 3'-5' junctions of the circularized mRNAs found the intra-molecular ligations occurred between fully processed 5' ends (the start AUG) and a variable 3' terminus. For five genes (cob, cox, nd2, nd4, and nd6), some of the 3' ends maintained an oligonucleotide addition during ligation, and for two of them, cob and nd6, these 3' termini were the most commonly recovered sequence. Previous reports have shown that after cleavage, three untemplated oligonucleotide additions may occur on the 3' termini of these mRNAs-adenylation, uridylylation, or cytidylation. These results suggest oligo(U) and oligo(C) additions may be part of the maturation process since they are maintained in the circular mRNAs. Circular RNAs occur in organisms across the biological spectrum, but their purpose in some systems, such as organelles (mitochondria and chloroplasts) is unclear. We hypothesize, that in C. reinhardtii mitochondria it may create a leader sequence to facilitate translation initiation, which may negate the need for an alternative translation initiation mechanism in this system, as previously speculated. In addition, circularization may play a protective role against exonucleases, and/or increase translational productivity.

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The non-pathogenic bacterium Mycobacterium smegmatis mc2155 has been widely used as a model organism in mycobacterial research, yet a detailed study about its transcription landscape remains to be established. Here we report the transcriptome, expression profiles and transcriptional structures through growth-phase-dependent RNA sequencing (RNA-seq) as well as other related experiments. We found: (1) 2,139 transcriptional start sites (TSSs) in the genome-wide scale, of which eight samples were randomly selected and further verified by 5'-RACE; (2) 2,233 independent monocistronic or polycistronic mRNAs in the transcriptome within the operon/sub-operon structures which are classified into five groups; (3) 47.50% (1016/2139) genes were transcribed into leaderless mRNAs, with the TSSs of 41.3% (883/2139) mRNAs overlapping with the first base of the annotated start codon. Initial amino acids of MSMEG_4921 and MSMEG_6422 proteins were identified by Edman degradation, indicating the presence of distinctive widespread leaderless features in M. smegmatis mc2155. (4) 150 genes with potentially wrong structural annotation, of which 124 proposed genes have been corrected; (5) eight highly active promoters, with their activities further determined by ß-galactosidase assays. These data integrated the transcriptional landscape to genome information of model organism mc2155 and lay a solid foundation for further works in Mycobacterium.

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A strategy that can be applied to the research of new molecules with antibacterial activity is to look for inhibitors of essential bacterial processes within large collections of chemically heterogeneous compounds. The implementation of this approach requires the development of proper assays aimed at the identification of molecules interfering with specific cell pathways and potentially applicable to the high throughput analysis of large chemical library. Here, I describe a fluorescence-based whole-cell assay in Escherichia coli devised to find inhibitors of the translation initiation pathway. Translation is a complex and essential mechanism. It involves numerous sub-steps performed by factors that are in many cases sufficiently dissimilar in bacterial and eukaryotic cells to be targetable with domain-specific drugs. As a matter of fact, translation has been proven as one of the few bacterial mechanisms pharmacologically tractable with specific antibiotics. The assay described in this chapter is tailored to the identification of molecules affecting the first stage of translation initiation, which is the most dissimilar step in bacteria vs. mammals. The effect of the compounds under analysis is assayed in living cells, thus allowing evaluating their in vivo performance as inhibitors of translation initiation. Compared with other assays for antibacterials, the major advantages of this screen are its simplicity and high mechanism specificity.

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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.

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The bacterial translational apparatus is an ideal target for the search of new antibiotics. In fact, it performs an essential process carried out by a large number of potential subtargets for antibiotic action. Moreover, it is sufficiently different in several molecular details from the apparatus of Eukarya and Archaea to generally ensure specificity for the bacterial domain. This applies in particular to translation initiation, which is the most different step in the process. In bacteria, the 30S ribosomal subunit directly binds to the translation initiation region, a site within the messenger RNA (mRNA) 5'-untranslated region (5'-UTR). 30S binding is mediated by the interaction of both the 16S ribosomal RNA and the ribosomal protein S1 with specific regions of the mRNA 5'-UTR. An alternative, S1-independent pathway is enjoyed by leaderless mRNAs (i.e., transcripts devoid of a 5'-UTR). We have developed a simple fluorescence-based whole-cell assay in Escherichia coli to find inhibitors of the canonical S1-dependent translation initiation pathway. The assay has been set up both in a common E. coli laboratory strain and in a strain with an outer membrane permeability defect. Compared with other whole-cell assays for antibacterials, the major advantages of the screen described here are high sensitivity and specificity.

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