Determination of ribonuclease sequence-specificity using Pentaprobes and mass spectrometry

McKenzie, Joanna Leigh; Duyvestyn, Johanna Maria; Smith, Tony C.; Bendak, Katerina; Mackay, Joel P.; Cursons, Raymond T.; Cook, Gregory M.; Arcus, Vickery L.

doi:10.1261/rna.031229.111

Cited by 40 publications

(55 citation statements)

References 45 publications

(54 reference statements)

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“…15 and this work), other prokaryotic VapCs exhibit nonspecific RNase activity in vitro 9,31-34 or cleave degenerated recognition motifs in synthetic RNAs 35,36 . Further, in one case (VapC4 of M. tuberculosis), the presumed PIN-domain ribonuclease binds in vitro to RNAs containing the motif ACGC and is suggested to inhibit translation by stoichiometric binding to mRNAs containing such recognition motifs 20 .…”

Section: Discussionmentioning

confidence: 71%

VapC20 of Mycobacterium tuberculosis cleaves the Sarcin–Ricin loop of 23S rRNA

et al. 2013

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The highly persistent and often lethal human pathogen, Mycobacterium tuberculosis contains at least 88 toxin-antitoxin genes. More than half of these encode VapC PIN domain endoribonucleases that inhibit cell growth by unknown mechanisms. Here we show that VapC20 of M. tuberculosis inhibits translation by cleavage of the Sarcin-Ricin loop (SRL) of 23S ribosomal RNA at the same position where Sarcin and other eukaryotic ribotoxins cleave. Toxin-inhibited cells can be rescued by the expression of the antitoxin, thereby raising the possibility that vapC20 contributes to the extreme persistence exhibited by M. tuberculosis. VapC20 cleavage is inhibited by mutations in the SRL that flank the cleavage site but not by changes elsewhere in the loop. Disruption of the SRL stem abolishes cleavage; however, further mutations that restore the SRL stem structure restore cleavage, revealing that the structure rather than the exact sequence of the SRL is important for this activity.

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Section: Discussionmentioning

confidence: 71%

VapC20 of Mycobacterium tuberculosis cleaves the Sarcin–Ricin loop of 23S rRNA

et al. 2013

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“…The functions of a number of VapC toxins from Mycobacterium tuberculosis have been reported. VapC20 (Rv2549c) cleaves 23S rRNA, while VapC1 (Rv0065) and VapC29 (Rv0617) cut single-stranded RNAs in GC-rich sequences (23,24) and VapC4 (Rv0595c) appears to inhibit translation by binding to mRNAs (25).…”

mentioning

confidence: 99%

Structure-Function Analysis of VapB4 Antitoxin Identifies Critical Features of a Minimal VapC4 Toxin-Binding Module

2015

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T oxin-antitoxin (TA) systems were first identified to be addictive genetic elements responsible for the maintenance of plasmid stability during cell segregation (1, 2). Subsequently, chromosomally encoded TA systems were characterized to be involved in several mechanisms of toxin action (3, 4). TA systems typically consist of two elements, a toxin protein that suppresses cell growth by targeting essential cellular metabolic pathways and an antitoxin RNA or protein that binds to and blocks the growth inhibition activity of the toxin. Different types of TA systems have been defined according to the nature and modes of action of antitoxins, and of these, the type II TA systems are the most abundant and well studied (5, 6). The type II toxins-antitoxins are typically encoded in the same operon, where the gene for the antitoxin is located upstream of that for the toxin, except in the case of higBA, which has a reversed gene order (7). The type II antitoxin is a protein that directly interacts with its cognate toxin, resulting in neutralization of toxicity. The antitoxin or the toxin-antitoxin protein complex often acts as a repressor that autoregulates the transcription of TA operons via its ability to bind to the promoter (8). During cellular stress, the antitoxin is hydrolyzed by a protease, such as Lon or Clp, to release the toxin from the protein complex and derepress transcription of the TA operon (7). These properties allow TA systems to play regulatory roles in bacterial gene expression and the establishment and maintenance of dormancy. This nonreplicative state may contribute to latency.Type II TA systems are classified into several different families on the basis of sequence similarities and the functions of the toxins (7-9). CcdB and ParE inhibit DNA replication by targeting DNA gyrase (10, 11). Doc arrests translation elongation by phosphorylation of elongation factor Tu (12, 13). Kis and HicA cleave mRNA in a ribosome-independent manner (14, 15). MazF targets various RNAs, including mRNA, 16S rRNA,. RelE inhibits translation by cleavage of ribosome-bound mRNAs at the ribosomal A site (20). HipA inhibits translation by phosphoryla-

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“…RNase T, the substrate binding site from one protomer of the dimer and the catalytic site from the other together form the functional RNase (23). The overall orientation of the protomers is similar in Rv2179c, suggesting a similar substrate binding mode involving both protomers (Fig.…”

Section: The Rv2179c Substrate Binding Site Is Distinct-in E Colimentioning

confidence: 92%

Mycobacterium tuberculosis Rv2179c Protein Establishes a New Exoribonuclease Family with Broad Phylogenetic Distribution

Abendroth¹,

Ollodart

Andrews

et al. 2014

Journal of Biological Chemistry

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Background: More than 25% of Mtb proteins have unknown functions, limiting our understanding of Mtb physiology and pathogenesis. Results: The hypothetical Mtb protein Rv2179c is a new DEDD-type exoribonuclease. Conclusion: Rv2179c is the founding member of a new exoribonuclease family with broad phylogenetic distribution. Significance: Assigning RNase function to Rv2179c provides annotation for hundreds of bacterial orthologs and provides a new view of RNA processing in Mtb.

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Determination of ribonuclease sequence-specificity using Pentaprobes and mass spectrometry

Cited by 40 publications

References 45 publications

VapC20 of Mycobacterium tuberculosis cleaves the Sarcin–Ricin loop of 23S rRNA

VapC20 of Mycobacterium tuberculosis cleaves the Sarcin–Ricin loop of 23S rRNA

Structure-Function Analysis of VapB4 Antitoxin Identifies Critical Features of a Minimal VapC4 Toxin-Binding Module

Mycobacterium tuberculosis Rv2179c Protein Establishes a New Exoribonuclease Family with Broad Phylogenetic Distribution

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