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1.
Nat Commun ; 12(1): 4909, 2021 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-34389707

RESUMO

In bacteria, trans-translation is the main rescue system, freeing ribosomes stalled on defective messenger RNAs. This mechanism is driven by small protein B (SmpB) and transfer-messenger RNA (tmRNA), a hybrid RNA known to have both a tRNA-like and an mRNA-like domain. Here we present four cryo-EM structures of the ribosome during trans-translation at resolutions from 3.0 to 3.4 Å. These include the high-resolution structure of the whole pre-accommodated state, as well as structures of the accommodated state, the translocated state, and a translocation intermediate. Together, they shed light on the movements of the tmRNA-SmpB complex in the ribosome, from its delivery by the elongation factor EF-Tu to its passage through the ribosomal A and P sites after the opening of the B1 bridges. Additionally, we describe the interactions between the tmRNA-SmpB complex and the ribosome. These explain why the process does not interfere with canonical translation.


Assuntos
Proteínas de Escherichia coli/genética , Escherichia coli/genética , Biossíntese de Proteínas/genética , RNA Bacteriano/genética , Proteínas de Ligação a RNA/genética , Ribossomos/genética , Sítios de Ligação/genética , Microscopia Crioeletrônica , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Modelos Moleculares , Conformação de Ácido Nucleico , Ligação Proteica , Domínios Proteicos , RNA Bacteriano/química , RNA Bacteriano/metabolismo , RNA Mensageiro/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA de Transferência/química , RNA de Transferência/genética , RNA de Transferência/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Ribossomos/metabolismo , Ribossomos/ultraestrutura
2.
Nucleic Acids Res ; 48(4): e22, 2020 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-31919515

RESUMO

In order to discover new antibiotics with improved activity and selectivity, we created a reliable in vitro reporter system to detect trans-translation activity, the main mechanism for recycling ribosomes stalled on problematic messenger RNA (mRNA) in bacteria. This system is based on an engineered tmRNA variant that reassembles the green fluorescent protein (GFP) when trans-translation is active. Our system is adapted for high-throughput screening of chemical compounds by fluorescence.


Assuntos
Bactérias/genética , Proteínas de Fluorescência Verde/genética , Biossíntese de Proteínas , RNA Mensageiro/genética , Antibacterianos/síntese química , Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Humanos , Biossíntese de Proteínas/efeitos dos fármacos , Proteínas de Ligação a RNA/genética , Ribossomos/efeitos dos fármacos , Ribossomos/genética
3.
Ann N Y Acad Sci ; 1447(1): 80-87, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30815901

RESUMO

In bacteria, trans-translation is the primary quality control mechanism for rescuing ribosomes arrested during translation. This key process is universally conserved and plays a crucial role in the viability and virulence of all bacteria. It is performed by transfer-messenger RNA (tmRNA) and its protein partner small protein B (SmpB). Here, we show that tmRNA is a key molecule that could have given birth to modern protein synthesis. The traces of an ancient RNA world persist in the structure of modern tmRNA, suggesting its old origins. Therefore, since it has both tRNA and mRNA functions, tmRNA could be the missing link that allowed modern genetic code to be read by the ribosome.


Assuntos
Biossíntese de Proteínas/fisiologia , RNA Bacteriano/genética , RNA Mensageiro/genética , RNA de Transferência/genética , Animais , Humanos , Estrutura Secundária de Proteína , RNA Bacteriano/química , RNA Mensageiro/química , RNA de Transferência/química
4.
Nat Commun ; 10(1): 752, 2019 02 14.
Artigo em Inglês | MEDLINE | ID: mdl-30765709

RESUMO

Viruses modulate ecosystems by directly altering host metabolisms through auxiliary metabolic genes. However, viral genomes are not known to encode the core components of translation machinery, such as ribosomal proteins (RPs). Here, using reference genomes and global-scale viral metagenomic datasets, we identify 14 different RPs across viral genomes arising from cultivated viral isolates and metagenome-assembled viruses. Viruses tend to encode dynamic RPs, easily exchangeable between ribosomes, suggesting these proteins can replace cellular versions in host ribosomes. Functional assays confirm that the two most common virus-encoded RPs, bS21 and bL12, are incorporated into 70S ribosomes when expressed in Escherichia coli. Ecological distribution of virus-encoded RPs suggests some level of ecosystem adaptations as aquatic viruses and viruses of animal-associated bacteria are enriched for different subsets of RPs. Finally, RP genes are under purifying selection and thus likely retained an important function after being horizontally transferred into virus genomes.

5.
J Mol Biol ; 429(23): 3617-3625, 2017 11 24.
Artigo em Inglês | MEDLINE | ID: mdl-29031699

RESUMO

In bacteria, trans-translation is the main quality control mechanism for rescuing ribosomes arrested during translation. This key process is universally conserved and plays a critical role in the viability and virulence of many pathogens. We developed a reliable in vivo double-fluorescence reporter system for the simultaneous quantification of both trans-translation and the associated proteolysis activities in bacteria. The assay was validated using mutant bacteria lacking tmRNA, SmpB, and the ClpP protease. Both antisense tmRNA-binding RNA and a peptide mimicking the SmpB C-terminal tail proved to be potent inhibitors of trans-translation in vivo. The double-fluorescent reporter was also tested with KKL-35, an oxadiazole derivative that is supposed to be a promising trans-translation inhibitor, and it surprisingly turns out that trans-translation is not the only target of KKL-35 in vivo.


Assuntos
Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Biossíntese de Proteínas , RNA Bacteriano/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ribossomos/metabolismo , Proteínas de Bactérias/genética , RNA Bacteriano/genética , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Ribossomos/genética
6.
Extremophiles ; 20(3): 301-10, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-27039108

RESUMO

Bacteria of the genus Photobacterium thrive worldwide in oceans and show substantial eco-physiological diversity including free-living, symbiotic and piezophilic life styles. Genomic characteristics underlying this variability across species are poorly understood. Here we carried out genomic and physiological analysis of Photobacterium phosphoreum strain ANT-2200, the first deep-sea luminous bacterium of which the genome has been sequenced. Using optical mapping we updated the genomic data and reassembled it into two chromosomes and a large plasmid. Genomic analysis revealed a versatile energy metabolic potential and physiological analysis confirmed its growth capacity by deriving energy from fermentation of glucose or maltose, by respiration with formate as electron donor and trimethlyamine N-oxide (TMAO), nitrate or fumarate as electron acceptors, or by chemo-organo-heterotrophic growth in rich media. Despite that it was isolated at a site with saturated dissolved oxygen, the ANT-2200 strain possesses four gene clusters coding for typical anaerobic enzymes, the TMAO reductases. Elevated hydrostatic pressure enhances the TMAO reductase activity, mainly due to the increase of isoenzyme TorA1. The high copy number of the TMAO reductase isoenzymes and pressure-enhanced activity might imply a strategy developed by bacteria to adapt to deep-sea habitats where the instant TMAO availability may increase with depth.


Assuntos
Adaptação Fisiológica , Metabolismo Energético , Genoma Bacteriano , Photobacterium/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Transporte de Elétrons , Glucose/metabolismo , Pressão Hidrostática , Isoenzimas/genética , Isoenzimas/metabolismo , Maltose/metabolismo , Metilaminas/metabolismo , Oxirredutases N-Desmetilantes/genética , Oxirredutases N-Desmetilantes/metabolismo , Photobacterium/metabolismo , Água do Mar/microbiologia
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