Characterization of Escherichia coli dinJ-yafQ toxin-antitoxin system using insights from mutagenesis data

Escherichia coli dinJ-yafQ operon codes for a functional toxin-antitoxin (TA) system. YafQ toxin is an RNase which, upon overproduction, specifically inhibits the translation process by cleaving cellular mRNA at specific sequences. DinJ is an antitoxin and counteracts YafQ-mediated toxicity by forming a strong protein complex. In the present study we used site-directed mutagenesis of YafQ to determine the amino acids important for its catalytic activity. His50Ala, His63Ala, Asp67Ala, Trp68Ala, Trp68Phe, Arg83Ala, His87Ala, and Phe91Ala substitutions of the predicted active-site residues of YafQ abolished mRNA cleavage in vivo, whereas Asp61Ala and Phe91Tyr mutations inhibited YafQ RNase activity only moderately. We show that YafQ, upon overexpression, cleaved mRNAs preferably 5' to A between the second and third nucleotides in the codon in vivo. YafQ also showed RNase activity against mRNA, tRNA, and 5S rRNA molecules in vitro, albeit with no strong specificity. The endoribonuclease activity of YafQ was inhibited in the complex with DinJ antitoxin in vitro. DinJ-YafQ protein complex and DinJ antitoxin alone selectively bind to one of the two palindromic sequences present in the intergenic region upstream of the dinJ-yafQ operon, suggesting the autoregulation mode of this TA system.     

Molecular basis of ribosome recognition and mRNA hydrolysis by the E. coli YafQ toxin

Bacterial type II toxin-antitoxin modules are protein–protein complexes whose functions are finely tuned by rapidly changing environmental conditions. E. coli toxin YafQ is suppressed under steady state growth conditions by virtue of its interaction with its cognate antitoxin, DinJ. During stress, DinJ is proteolytically degraded and free YafQ halts translation by degrading ribosome-bound mRNA to slow growth until the stress has passed. Although structures of the ribosome with toxins RelE and YoeB have been solved, it is unclear what residues among ribosome-dependent toxins are essential for mediating both recognition of the ribosome and the mRNA substrate given their low sequence identities. Here we show that YafQ coordinates binding to the 70S ribosome via three surface-exposed patches of basic residues that we propose directly interact with 16S rRNA. We demonstrate that YafQ residues H50, H63, D67 and H87 participate in acid-base catalysis during mRNA hydrolysis and further show that H50 and H63 functionally complement as general bases to initiate the phosphodiester cleavage reaction. Moreover YafQ residue F91 likely plays an important role in mRNA positioning. In summary, our findings demonstrate the plasticity of ribosome-dependent toxin active site residues and further our understanding of which toxin residues are important for function.     

Escherichia coli dinJ-yafQ genes act as a toxin-antitoxin module

Bacterial toxin-antitoxin (TA) systems are operons that code for a stable toxic protein and a labile antitoxin. TA modules are widespread on the chromosomes of free-living Bacteria and Archaea, where they presumably act as stress response elements. The chromosome of Escherichia coli K-12 encodes four known TA pairs, as well as the dinJ-yafQ operon, which is hypothesized to be a TA module based on operon organization similar to known TA genes. Induction of YafQ inhibited cell growth, but its toxicity was counteracted by coexpression of dinJ cloned on a separate plasmid. YafQ(His)(6) and DinJ proteins coeluted in Ni(2+)-affinity and gel filtration chromatography, implying the formation of a specific and stable YafQ-DinJ protein complex with an estimated molecular mass of c. 37.3 kDa. Induction of YafQ reduced protein synthesis up to 40% as judged by incorporation of [(35)S]-methionine, but did not influence the rates of DNA and RNA synthesis. Structure modelling of E. coli YafQ revealed its structural relationship with bacterial toxins of known structure suggesting that it might act as a sequence-specific mRNA endoribonuclease.     

Genetic Diversity in Exon 2 of the Major Histocompatibility Complex Class II DQB1 Locus in Nigerian Goats

The DQB1 locus is located in the major histocompatibility complex (MHC) class II region and involved in immune response. We identified 20 polymorphic sites in a 228 bp fragment of exon 2, one of the most critical regions of the MHC DQB1 gene, in 60 Nigerian goats. Four sites are located in the peptide binding region, and 10 amino acid substitutions are peculiar to Nigerian goats, compared with published sequences. A significantly higher ratio of nonsynonymous/synonymous substitutions (d _N/d _S) suggests that allelic sequence evolution is driven by balancing selection (P < 0.01). In silico functional analysis using PANTHER predicted that substitution P56R, with a subPSEC score of ?4.00629 (P_deleterious = 0.73229), is harmful to protein function. The phylogenetic tree from consensus sequences placed the two northern breeds closer to each other than either was to the southern goats. This first report of sequence diversity at the DQB1 locus for any African goat breed may be useful in the search for disease-resistant genotypes.