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Reconstitution of Protein Translation of Mycobacterium Reveals Functional Conservation and Divergence with the Gram-Negative Bacterium Escherichia coli.

ABSTRACT: Protein translation is essential for all bacteria pathogens. It has also been a major focus of structural and functional studies and an important target of antibiotics. Here we report our attempts to biochemically reconstitute mycobacterial protein translation in vitro from purified components. This mycobacterial translation system consists of individually purified recombinant translation factors from Mycobacterium tuberculosis (M. tuberculosis), purified tRNAs and ribosomes from Mycobacterium smegmatis (M. smegmatis), and an aminoacyl-tRNA synthetase (AARS) mixture from the cell-extract of M. smegmatis. We demonstrate that such mycobacterial translation system was efficient in in vitro protein synthesis, and enabled functional comparisons of translational components between the gram-positive Mycobacterium and the gram-negative E. coli. Although mycobacterial translation factors and ribosomes were highly compatible with their E. coli counterparts, M. smegmatis tRNAs were not properly charged by the E. coli AARSs to allow efficient translation of a reporter. In contrast, both E. coli and M. smegmatis tRNAs exhibited similar activity with the semi-purified M. smegmatis AARSs mixture for in vitro translation. We further demonstrated the use of both mycobacterial and E. coli translation systems as comparative in vitro assays for small-molecule antibiotics that target protein translation. While mycobacterial and E. coli translation were both inhibited at the same IC50 by the antibiotic spectinomycin, mycobacterial translation was preferentially inhibited by the antibiotic tetracycline, suggesting that there may be structural differences at the antibiotic binding sites between the ribosomes of Mycobacterium and E. coli. Our results illustrate an alternative approach for antibiotic discovery and functional studies of protein translation in mycobacteria and possibly other bacterial pathogens.

SUBMITTER: Srivastava A

PROVIDER: S-EPMC5001721 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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Reconstitution of Protein Translation of Mycobacterium Reveals Functional Conservation and Divergence with the Gram-Negative Bacterium Escherichia coli.

Srivastava Aashish A Asahara Haruichi H Zhang Meng M Zhang Weijia W Liu Haiying H Cui Sheng S Jin Qi Q Chong Shaorong S

PloS one 20160826 8

Protein translation is essential for all bacteria pathogens. It has also been a major focus of structural and functional studies and an important target of antibiotics. Here we report our attempts to biochemically reconstitute mycobacterial protein translation in vitro from purified components. This mycobacterial translation system consists of individually purified recombinant translation factors from Mycobacterium tuberculosis (M. tuberculosis), purified tRNAs and ribosomes from Mycobacterium s ...[more]

PMID: 27564552

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Project description:BackgroundTrans-translation is catalyzed by ribonucleprotein complexes composed of SmpB protein and transfer-messenger RNA. They release stalled ribosomes from truncated mRNAs and tag defective proteins for proteolytic degradation. Comparative sequence analysis of bacterial tmRNAs provides considerable insights into their secondary structures in which a tRNA-like domain and an mRNA-like region are connected by a variable number of pseudoknots. Progress toward understanding the molecular mechanism of trans-translation is hampered by our limited knowledge about the structure of tmRNA:SmpB complexes.ResultsComplexes consisting of M. tuberculosis tmRNA and E. coli SmpB tag truncated proteins poorly in E. coli. In contrast, the tagging activity of E. coli tmRNA is well supported by M. tuberculosis SmpB that is expressed in E. coli. To investigate this incompatibility, we constructed 12 chimeric tmRNA molecules composed of structural features derived from both E. coli and M. tuberculosis. Our studies demonstrate that replacing the hp5-pk2-pk3-pk4 segment of E. coli tmRNA with the equivalent segment of M. tuberculosis tmRNA has no significant effect on the tagging efficiency of chimeric tmRNAs in the presence of E. coli SmpB. Replacing either helices 2b-2d, the single-stranded part of the ORF, pk1, or residues 79-89 of E. coli tmRNA with the equivalent features of M. tuberculosis tmRNA yields chimeric tmRNAs that are tagged at 68 to 88 percent of what is observed with E. coli tmRNA. Exchanging segments composed of either pk1 and the single-stranded segment upstream of the ORF or helices 2b-2d and pk1 results in markedly impaired tagging activity.ConclusionOur observations demonstrate the existence of functionally important but as yet uncharacterized structural constraints in the segment of tmRNA that connects its TLD to the ORF used for resuming translation. As trans-translation is important for the survival of M. tuberculosis, our work provides a new target for pharmacological intervention against multidrug-resistant tuberculosis.

Dataset Information

Reconstitution of Protein Translation of Mycobacterium Reveals Functional Conservation and Divergence with the Gram-Negative Bacterium Escherichia coli.

Publications

Reconstitution of Protein Translation of Mycobacterium Reveals Functional Conservation and Divergence with the Gram-Negative Bacterium Escherichia coli.

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