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

Promoter-sequence determinants and structural basis of primer-dependent transcription initiation in Escherichia coli

Chemical modifications of RNA 5'-ends enable "epitranscriptomic" regulation, influencing multiple aspects of RNA fate. In transcription initiation, a large inventory of substrates compete with nucleoside triphosphates for use as initiating entities, providing an ab initio mechanism for altering the RNA 5'-end. In Escherichia coli cells, RNAs with a 5'-end hydroxyl are generated by use of dinucleotide RNAs as primers for transcription initiation, "primer-dependent initiation." Here, we use massively systematic transcript end readout (MASTER) to detect and quantify RNA 5'-ends generated by primer-dependent initiation for ∼410 (∼1,000,000) promoter sequences in E. coli The results show primer-dependent initiation in E. coli involves any of the 16 possible dinucleotide primers and depends on promoter sequences in, upstream, and downstream of the primer binding site. The results yield a consensus sequence for primer-dependent initiation, YTSS-2NTSS-1NTSSWTSS+1, where TSS is the transcription start site, NTSS-1NTSS is the primer binding site, Y is pyrimidine, and W is A or T. Biochemical and structure-determination studies show that the base pair (nontemplate-strand base:template-strand base) immediately upstream of the primer binding site (Y:RTSS-2, where R is purine) exerts its effect through the base on the DNA template strand (RTSS-2) through interchain base stacking with the RNA primer. Results from analysis of a large set of natural, chromosomally encoded E coli promoters support the conclusions from MASTER. Our findings provide a mechanistic and structural description of how TSS-region sequence hard-codes not only the TSS position but also the potential for epitranscriptomic regulation through primer-dependent transcription initiation

Ubiquitous Promoter-Localization of Essential Virulence Regulators in Francisella tularensis

Francisella tularensis is a Gram-negative bacterium whose ability to replicate within macrophages and cause disease is strictly dependent upon the coordinate activities of three transcription regulators called MglA, SspA, and PigR. MglA and SspA form a complex that associates with RNA polymerase (RNAP), whereas PigR is a putative DNA-binding protein that functions by contacting the MglA-SspA complex. Most transcription activators that bind the DNA are thought to occupy only those promoters whose activities they regulate. Here we show using chromatin immunoprecipitation coupled with high-throughput DNA sequencing (ChIP-Seq) that PigR, MglA, and SspA are found at virtually all promoters in F. tularensis and not just those of regulated genes. Furthermore, we find that the ability of PigR to associate with promoters is dependent upon the presence of MglA, suggesting that interaction with the RNAP-associated MglA-SspA complex is what directs PigR to promoters in F. tularensis. Finally, we present evidence that the ability of PigR (and thus MglA and SspA) to positively control the expression of genes is dictated by a specific 7 base pair sequence element that is present in the promoters of regulated genes. The three principal regulators of virulence gene expression in F. tularensis therefore function in a non-classical manner with PigR interacting with the RNAP-associated MglA-SspA complex at the majority of promoters but only activating transcription from those that contain a specific sequence element. Our findings reveal how transcription factors can exert regulatory effects at a restricted set of promoters despite being associated with most or all. This distinction between occupancy and regulatory effect uncovered by our data may be relevant to the study of RNAP-associated transcription regulators in other pathogenic bacteria

MglA, SspA, and PigR are found ubiquitously at promoter regions.

<p>A representative illustration of the density of the normalized mapped sequencing reads after ChIP-Seq of β′+rif (purple), σ⁷⁰ (orange), MglA (brown), SspA (light pink), PigR (blue), and HipB (dark pink) (A) at the FTL_0491 promoter, which is known to be regulated by MglA, SspA, and PigR; (normalized reads are displayed on a linear scale) (B) at the FTL_0650 and FTL_0651 promoters, which are not under the control of MglA, SspA, or PigR (normalized reads are displayed on a linear scale); and (C) across a 400 kb region of the <i>F</i>. <i>tularensis</i> chromosome (normalized reads are displayed on a log scale). There is significant concordance between the enrichment profiles of β′+rif, σ⁷⁰, MglA, SspA, and PigR. HipB is not specifically enriched at these regions. (D) Venn diagram representing the overlap between MglA, SspA, and PigR peaks at σ⁷⁰-associated promoters. Numbers indicate percent of promoters that are enriched for the indicated transcription factor.</p

PigR requires MglA to specifically associate with promoter regions.

<p>(A) Abundance of ectopically expressed PigR-V as analyzed by Western blot. (<i>Upper</i>) Western blot probed with antibody against the VSV-G tag. (<i>Lower</i>) Western blot probed with antibody against GroEL serves as a loading control. Wild-type LVS cells containing the empty control vector pF (lane 1); LVS ∆<i>pigR</i> mutant cells containing either pF-PigR-V (lane 2) or pF2-PigR-V (lane 3); LVS ∆<i>pigR</i> ∆<i>mglA</i> mutant cells containing pF-PigR-V (lane 4). (B) and (C) Representative datasets illustrating the density of the normalized mapped sequencing reads after ChIP-Seq with cells of the LVS σ⁷⁰-V strain (orange), cells of the LVS PigR-V strain (blue), cells of the LVS ∆<i>pigR</i> mutant strain that ectopically synthesize PigR-V from plasmid pF2-PigR-V (PigR^e ∆<i>pigR</i>, light blue), and cells of the LVS ∆<i>pigR</i> ∆<i>mglA</i> mutant strain that ectopically synthesize PigR-V from plasmid pF-PigR-V (PigR^e ∆<i>pigR</i> ∆<i>mglA</i>, blue-green). (B) Ectopically produced PigR-V occupies the promoter of the PigR/MglA/SspA-regulated FTL_0491 gene in cells containing MglA, but not in cells lacking MglA. (C) Ectopically produced PigR-V occupies the promoters of the FTL_0650 and FTL_0651 genes, which are not under the control of PigR/MglA/SspA, only in cells that contain MglA.</p

Identification of promoters in <i>F</i>. <i>tularensis</i> using ChIP-Seq.

<p>(A) Schematic representation of the VSV-G tag integration vector and its use to construct the LVS β′-V strain that synthesizes the β′ subunit of RNAP with a VSV-G tag (β′-V) at native levels. (B) A representative illustration of the density of normalized mapped sequencing reads (Y-axis) along a region of the chromosome (X-axis) after ChIP-Seq of each epitope-tagged factor: β′ (green), β′+ rif (purple), σ⁷⁰ (orange), σ³² (cyan), and HipB (dark pink). Gray boxes below the read density plot indicate areas of significantly enriched reads; red lines indicate sites of maximum enrichment. Promoter regions, defined as areas with significant enrichment of β′ + rif, σ³², or σ⁷⁰ with the chromosome, are indicated by the purple boxes below the gene annotations.</p

The PigR response element (PRE) is necessary and sufficient for promoters to be controlled by PigR.

<p>(A) A logo of the 7 bp consensus PRE sequence motif generated by MEME. (B) Alignment of promoters with mapped transcription start sites and predicted -10 and -35 elements (underlined), including the promoters of five PigR-regulated genes (FTL_0026, <i>iglA pdpA</i>, FTL_1218, FTL_1219, FTL_0361) and the promoters of three non-PigR regulated genes (FTL_0361, <i>pmrA</i>, <i>hupB</i>). The conserved PRE is found 6–7 bp upstream of the -35 element only in those promoters known to be controlled by PigR. (C) Alignment of <i>iglA</i> promoter variants. Nucleotide substitutions (in bold) were introduced in the <i>iglA</i> promoter fused to the <i>lacZ</i> reporter gene and integrated into the FTL_0111 locus. (D) Quantification of <i>lacZ</i> expression in strains LVS and LVS ∆<i>pigR</i> containing the indicated promoter variants (indicated along the X-axis) by β-galactosidase assay, as measured in Miller Units (Y-axis). (E) Alignment of FTL_0361 promoter variants. Nucleotide substitutions (in bold) were introduced into the FTL_0361 promoter fused to the <i>lacZ</i> reporter gene and integrated into the FTL_0361 locus. (F) Quantification of <i>lacZ</i> expression in strains LVS and LVS ∆<i>pigR</i> containing the indicated promoter variants (indicated along the X-axis) by β-galactosidase assay, as measured in Miller Units (Y-axis). Error bars for the -10 mutant in the LVS strain, the wild type FTL_0361 promoter and the -10 mutant in the LVS ∆<i>pigR</i> strain are too small to be illustrated.</p

Model for how PigR functions coordinately with the MglA-SspA complex to positively control the expression of genes.

<p>PigR associates with RNAP through interaction with the RNAP-associated MglA-SspA complex and is consequently found associated with RNAP at the promoters of both non-regulated (A) and regulated (B) genes. (B) PigR is a DNA-binding protein that binds the PRE present within the promoters of regulated genes; contact between RNAP-bound PigR and the DNA stabilizes the binding of RNAP to the promoter, thereby activating transcription specifically from promoters that contain a PRE. Although for convenience, MglA and SspA are depicted here as interacting with the α subunit of RNAP, it is not known which subunit(s) of RNAP are contacted by the MglA-SspA complex.</p