tRNA is a new target for cleavage by a MazF toxin

Toxin-antitoxin (TA) systems play key roles in bacterial persistence, biofilm formation and stress responses. The MazF toxin from the Escherichia coli mazEF TA system is a sequence- and single-strand-specific endoribonuclease, and many studies have led to the proposal that MazF family members exclusively target mRNA. However, recent data indicate some MazF toxins can cleave specific sites within rRNA in concert with mRNA. In this report, we identified the repertoire of RNAs cleaved by Mycobacterium tuberculosis toxin MazF-mt9 using an RNA-seq-based approach. This analysis revealed that two tRNAs were the principal targets of MazF-mt9, and each was cleaved at a single site in either the tRNAPro14 D-loop or within the tRNALys43 anticodon. This highly selective target discrimination occurs through recognition of not only sequence but also structural determinants. Thus, MazF-mt9 represents the only MazF family member known to target tRNA and to require RNA structure for recognition and cleavage. Interestingly, the tRNase activity of MazF-mt9 mirrors basic features of eukaryotic tRNases that also generate stable tRNA-derived fragments that can inhibit translation in response to stress. Our data also suggest a role for tRNA distinct from its canonical adapter function in translation, as cleavage of tRNAs by MazF-mt9 downregulates bacterial growth

eLS - RNA Polymerases: Subunits and Functional Domains

RNA polymerase is a multisubunit enzyme that catalyses the synthesis of RNA. Although there is a striking degree of structural and functional conservation among all RNA polymerases, there are also many distinguishing features that reflect the specific requirements of individual organisms. Keywords: transcription; RNA polymerase; holoenzyme; subuni

Overview of Fic domain proteins, their targets, and their roles in bacterial virulence.

<p>The Fic domain is defined by a conserved structural fold composed of six to eight α-helices (red bar). A nine amino acid Fic motif (yellow box) within this core fold comprises the key residues for catalysis. Both the structure of the Fic domain and the sequence of the Fic motif informs the activity of the Fic domain protein. Important domains of the target proteins are shown as blue bars; modifications as yellow circles (not to scale). Known physiological manifestations of each class of Fic domain protein are illustrated below their target. The exact role of the NOI domains of RIN4 is not known, though they are exclusively represented in plant proteins and have been hypothesized to play a role in host response to pathogens <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004349#ppat.1004349-Afzal1" target="_blank">[32]</a>. Abbreviations on juggled balls: AMP, adenosine monophosphate; UMP, uridine monophosphate; P, phosphate; PC, phosphocholine.</p

Features and functions of effector proteins that contain a Fic domain.

<p>Fic proteins, their targets, and the residue(s) modified are shown. The far left column, running vertically, shows the modification type added by each Fic protein. Dashes, data not known.</p

Multiple Mechanisms of Suppression Circumvent Transcription Defects in an RNA Polymerase Mutant

Using a high-copy-number suppressor screen to obtain clues about the role of the yeast RNA polymerase II subunit RPB4 in transcription, we identified three suppressors of the temperature sensitivity resulting from deletion of the RPB4 gene (ΔRPB4). One suppressor is Sro9p, a protein related to La protein, another is the nucleosporin Nsp1p, and the third is the RNA polymerase II subunit RPB7. Suppression by RPB7 was anticipated since its interaction with RPB4 is well established both in vitro and in vivo. We examined the effect of overexpression of each suppressor gene on transcription. Interestingly, suppression of the temperature-sensitive phenotype correlates with the correction of a characteristic transcription defect of this mutant: each suppressor restored the level of promoter-specific, basal transcription to wild-type levels. Examination of the effects of the suppressors on other in vivo transcription aberrations in ΔRPB4 cells revealed significant amelioration of defects in certain inducible genes in Sro9p and RPB7, but not in Nsp1p, suppressor cells. Analysis of mRNA levels demonstrated that overexpression of each of the three suppressors minimally doubled the mRNA levels during stationary phase. However, the elevated mRNA levels in Sro9p suppressor cells appear to result from a combination of enhanced transcription and message stability. Taken together, these results demonstrate that these three proteins influence transcription and implicate Sro9p in both transcription and posttranscription events

Mycobacterium abscessus VapC5 toxin potentiates evasion of antibiotic killing by ribosome overproduction and activation of multiple resistance pathways

Abstract Mycobacterium abscessus (Mab) infections are inexplicably intractable to clearing after aggressive and lengthy treatment regimens. Here we discovered that acquisition of a single toxin-antitoxin system enables Mab to activate a phenotypic switch that enhances survival upon treatment with current first-line antibiotics. This switch is tripped when the VapC5 toxin inactivates tRNASerCGA by cleavage at only one site within its anticodon, leading to growth arrest. Concomitant tRNASerCGA depletion then reprograms the transcriptome to favor synthesis of proteins naturally low in the cognate Ser UCG codon including the transcription factor WhiB7 and members of its regulon as well as the ribosomal protein family. This programmed stockpiling of ribosomes is predicted to override the efficacy of ribosome-targeting antibiotics while the growth arrest phenotype attenuates antibiotics targeting cell wall synthesis. In agreement, VapC5 increases Mab persister formation upon exposure to amikacin and the next-generation oxazolidinone tedizolid (both target ribosomes) or cefoxitin (inhibits cell wall synthesis). These findings expand the repertoire of genetic adaptations harnessed by Mab to survive assaults intended to eradicate it, as well as provide a much-needed framework for selection of shorter and more efficacious alternate treatment options for Mab infections using currently available antimicrobials whose targets are not confounded by VapC5