E. coli MazF does not create specialized ribosomes that translate leaderless mRNAs, but instead blocks rRNA maturation and ribosome biogenesis [Ribo-Seq]
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ABSTRACT: E. coli MazF does not create specialized ribosomes that translate leaderless mRNAs, but instead blocks rRNA maturation and ribosome biogenesis [Ribo-Seq]
Project description:The purpose of this experiment was to characterize MazF cleavage specificity and locations of cleavage genome-wide in E. coli and also identify any leaderless transcripts .To do this, MazF was expressed in a ΔmazF background and compared to an empty vector. rRNA-subtracted samples were used to assess cleavage in the mRNA, and total RNA samples were used to assess changes in the rRNA.
Project description:E. coli MazF does not create specialized ribosomes that translate leaderless mRNAs, but instead blocks rRNA maturation and ribosome biogenesis
Project description:E. coli MazF does not create specialized ribosomes that translate leaderless mRNAs, but instead blocks rRNA maturation and ribosome biogenesis [RNA-seq]
Project description:E. coli MazF does not create specialized ribosomes that translate leaderless mRNAs, but instead blocks rRNA maturation and ribosome biogenesis [5'OH-seq]
Project description:The purpose of this experiment was to characterize MazF cleavage specificity with single-nucleotide resolution by enriching and sequencing 5’-OH ends (a 5’-OH end is generated by MazF cleavage).
Project description:The purpose of this experiment was to assess translation of mRNA after MazF expression to determine if cleavage events resulted in a loss in ribosome footprints.
Project description:Escherichia coli (E. coli) mazEF is an extensively studied stress-induced toxin-antitoxin (TA) system. The toxin MazF is an endoribonuclease that cleaves RNAs at ACA sites. Thereby, under stress the induced MazF generates a Stress induced Translation Machinery (STM), composed of MazF processed mRNAs and selective ribosomes that specifically translate the processed mRNAs. Here we performed a proteomic analysis of all the E. coli stress-induced proteins that are mediated through chromosomally borne mazF gene . We show that the mRNAs of almost all of them is characterized by the presence of an ACA site up to 100 nucleotides upstream to the AUG initiator. Thereby, under stressful conditions the induced MazF is processing mRNAs that are translated by STM. Furthermore, the presence of the ACA site far enough upstream (up to 100 nucleotides) to the AUG initiator may still permit translation by the canonical translation machinery. Thus, such dual translation mechanisms, enable under stress also to prepare proteins for immediate functions while coming back to normal growth conditions.
Project description:The universally conserved P-loop GTPases control diverse cellular processes, like signal transduction, ribosome assembly, cell motility, intracellular transport and translation. YchF belongs to the Obg-family of P-loop GTPases and is one of the least characterized member of this family. It is unique because it preferentially hydrolyses ATP rather than GTP, but its physiological role is largely unknown. Studies in different organisms including humans suggest a possible role of YchF in regulating the cellular adaptation to stress conditions. In the current study, we explored the role of YchF in the model organism Escherichia coli. By western blot and promotor fusion experiments, we demonstrate that the YchF levels decrease during stress conditions or when cells enter stationary phase. The decline in YchF levels trigger increased stress resistance and cells lacking YchF are resistant to multiple stress conditions, like oxidative stress, replication stress or translational stress. By in vivo site directed cross-linking we demonstrate that YchF interacts with the translation initiation factor IF3 and with multiple ribosomal proteins at the surface of the small ribosomal subunit. The absence of YchF enhances the anti-association activity of IF3, stimulates the translation of leaderless mRNAs and increases the resistance against the endoribonuclease MazF, which generates leaderless mRNAs during stress conditions. In summary, our data identify YchF as a stress-responsive regulator of leaderless mRNA translation
Project description:Synthetic circuits embedded in host-cells compete with cellular processes for limited intracellular resources. We show how funneling of cellular resources, after global transcriptome degradation by the sequence-dependent endoribonuclease MazF, to a synthetic circuit can increase production. Target genes are protected from MazF activity by recoding the gene sequence to eliminate recognition sites, while preserving the amino acid sequence. The expression of a protected fluorescent reporter and flux of a high-value metabolite are significantly enhanced using this genome-scale control strategy. Proteomics measurements discover a host factor in need of protection to improve resource redistribution activity. A computational model demonstrates that the MazF mRNA-decay feedback loop enables proportional control of MazF in an optimal operating regime. Transcriptional profiling of MazF-induced cells elucidates the dynamic shifts in transcript abundance and discovers regulatory design elements. Together, our results suggest that manipulation of cellular resource allocation is a key control parameter for synthetic circuit design.