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VapCs of Mycobacterium tuberculosis cleave RNAs essential for translation.

ABSTRACT: The major human pathogen Mycobacterium tuberculosis can survive in the host organism for decades without causing symptoms. A large cohort of Toxin-Antitoxin (TA) modules contribute to this persistence. Of these, 48 TA modules belong to the vapBC (virulence associated protein) gene family. VapC toxins are PIN domain endonucleases that, in enterobacteria, inhibit translation by site-specific cleavage of initiator tRNA. In contrast, VapC20 of M. tuberculosis inhibits translation by site-specific cleavage of the universally conserved Sarcin-Ricin loop (SRL) in 23S rRNA. Here we identify the cellular targets of 12 VapCs from M. tuberculosis by applying UV-crosslinking and deep sequencing. Remarkably, these VapCs are all endoribonucleases that cleave RNAs essential for decoding at the ribosomal A-site. Eleven VapCs cleave specific tRNAs while one exhibits SRL cleavage activity. These findings suggest that multiple vapBC modules contribute to the survival of M. tuberculosis in its human host by reducing the level of translation.

SUBMITTER: Winther K

PROVIDER: S-EPMC5175351 | biostudies-literature | 2016 Nov

REPOSITORIES: biostudies-literature

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VapCs of Mycobacterium tuberculosis cleave RNAs essential for translation.

Winther Kristoffer K Tree Jai J JJ Tollervey David D Gerdes Kenn K

Nucleic acids research 20160905 20

The major human pathogen Mycobacterium tuberculosis can survive in the host organism for decades without causing symptoms. A large cohort of Toxin-Antitoxin (TA) modules contribute to this persistence. Of these, 48 TA modules belong to the vapBC (virulence associated protein) gene family. VapC toxins are PIN domain endonucleases that, in enterobacteria, inhibit translation by site-specific cleavage of initiator tRNA. In contrast, VapC20 of M. tuberculosis inhibits translation by site-specific cl ...[more]

PMID: 27599842

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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.

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VapCs of Mycobacterium tuberculosis cleave RNAs essential for translation.

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VapCs of Mycobacterium tuberculosis cleave RNAs essential for translation.

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