Project description:The RNA-induced silencing complex, comprising Argonaute and guide RNA, mediates RNA interference. Here we report the 3.2 Å crystal structure of Kluyveromyces Argonaute (KpAGO) fortuitously complexed with guide RNA originating from small-RNA duplexes autonomously loaded by recombinant KpAGO. Despite their diverse sequences, guide-RNA nucleotides 1-8 are positioned similarly, with sequence-independent contacts to bases, phosphates and 2'-hydroxyl groups pre-organizing the backbone of nucleotides 2-8 in a near-A-form conformation. Compared with prokaryotic Argonautes, KpAGO has numerous surface-exposed insertion segments, with a cluster of conserved insertions repositioning the N domain to enable full propagation of guide-target pairing. Compared with Argonautes in inactive conformations, KpAGO has a hydrogen-bond network that stabilizes an expanded and repositioned loop, which inserts an invariant glutamate into the catalytic pocket. Mutation results and analogies to Ribonuclease H indicate that insertion of this glutamate finger completes a universally conserved catalytic tetrad, thereby activating Argonaute for cleavage. Employ high-throughput sequencing of small RNAs extracted from soluble or crystalline K. polysporus Ago1(207-1251) that had been purified from E. coli
Project description:This SuperSeries is composed of the following subset Series: GSE37723: Degradome sequencing from S. castellii GSE37724: Bacteria-derived small RNAs that co-purify with KpAGO Refer to individual Series
Project description:Type I interferons (IFNs) are consequential cytokines in antibacterial defense. Whether and how bacterial pathogens inhibit innate immune receptor-driven type I IFN expression remains mostly unknown. By screening a library of enterohemorrhagic Escherichia coli (EHEC) mutants, we uncovered EhaF, an uncharacterized protein, as an inhibitor of innate immune responses including IFNs. Further analyses identified EhaF as a secreted autotransporter—a type of bacterial secretion system with no known innate immune-modulatory function—that translocates into host cell cytosol and inhibit IFN response to EHEC. Mechanistically, EhaF interacts with and inhibits the MiT/TFE family transcription factor TFE3 resulting in impaired TANK phosphorylation and consequently, reduced IRF3 activation and type I IFN expression. Notably, EhaF-mediated innate immune suppression promotes EHEC colonization and pathogenesis in vivo. Overall, this study has uncovered a previously unknown autotransporter-based bacterial strategy that targets a specific transcription factor to subvert innate host defense.
Project description:Like many gram-negative phytopathogenic bacteria, Ralstonia solanacearum uses a type III secretion system to deliver into host cells a cocktail of effector proteins that can interfere with plant defences and promote infection. One of these effectors, the nuclear-targeted PopP2 acetyltransferase, was reported to inhibit many defensive WRKY transcription factors through acetylation. To gain a better understanding of the mechanisms by which PopP2 might exert its virulence functions, we searched for other PopP2-interacting partners. Here we report the identification of the Arabidopsis thaliana AT-Rich Interaction Domain protein 3 (ARID3) and its close homologs, ARID2 and ARID4 as additional targets of PopP2.
Project description:Whole genome sequencing was performed on E. coli BL21 (DE3) evolved at 25°C in pH 9 terrific broth media buffered with Tris-HCl (pH 9). The evolved E. coli was characterized and compared to the parent strain.
Project description:The only membrane-anchored and essential ATP-dependent protease in Escherichia coli is FtsH. It controls the intracellular concentration of the deacetylase LpxC, which catalyses the first committed step in lipopolysaccharide biosynthesis. LpxC stability is strictly regulated in a growth rate-dependent manner to ascertain a vital equilibrium of lipopolysaccharide (LPS) and phospholipid biosynthesis. Previous studies suggested the involvement of yet unknown factors in LpxC degradation. Aiming at the identification of such factors that are predicted to be associated with LpxC and/or FtsH at high and low growth rates, we established a quantitative super-SILAC LC-MS/MS-based approach. The identification of known LpxC and FtsH interactors validated our approach. Several enzymes involved in fatty acid biosynthesis and degradation, including the central regulator FadR, interacted with LpxC and/or FtsH and showed a significant impact on LpxC stability. The newly identified LpxC and FtsH interactor WaaH, a LPS-modifying enzyme, stimulates LpxC degradation. Our results go beyond the previously established link between LPS and phospholipid biosynthesis and uncover a far-reaching network that controls LPS biosynthesis by involving multiple enzymes in fatty acid metabolism and phospholipid biosynthesis and modification.
Project description:Bulk RNA-sequencing was performed on E. coli BL21 (DE3) evolved at 25°C in pH 9 terrific broth media buffered with Tris-HCl (pH 9). The evolved E. coli was characterised and compared to the parent strain during protein expression; the strains were actively grown and compared for gene expression at pH 7 and pH 9 in terrific broth media.
Project description:The genome of coronaviruses, including the SARS-CoV-2, which causes the human pandemic COVID-19, encodes for two proteases, a papain like (PL) and the so-called Main protease (Mpro), also named 3CLpro or non-structural protein 5 (NSP5). Mpro is activated by autoproteolysis, and true to its name, is the main protease responsible for cutting the viral polyprotein into functional viral proteins. Aside from this function, it has been described that 3CL proteases are also capable to process host proteins, including those involved in the host innate immune response. We performed a LC-MS based N-terminomics analysis to identify in vitro substrates of three different recombinantly expressed coronavirus main proteases (SARS-CoV, SARS-CoV-2, hCoV-NL63) using lung cell lysates as substrate pools.
Project description:Addressing the functionality of predicted genes remains an enormous challenge in the post-genomic era. A prime example of genes lacking functional assignments are the poorly conserved, early expressed genes of lytic bacteriophages, whose products are involved in the subversion of the host metabolism. In this study, we focused on the composition of important macromolecular complexes of Pseudomonas aeruginosa involved in transcription, DNA replication, fatty acid biosynthesis, RNA regulation, energy metabolism and cell division, during infection with members of seven distinct clades of lytic phages. Using affinity purifications of these host protein complexes coupled to mass spectrometric analyses, 37 host complex-associated phage proteins could be identified. Importantly, eight of these show an inhibitory effect on bacterial growth upon episomal expression, suggesting that these phage proteins are potentially involved in hijacking the host complexes. Using complementary protein-protein interaction assays, we further mapped the inhibitory interaction of gp12 of phage 14-1 to the α subunit of the RNA polymerase. Together, our data demonstrate the powerful use of interactomics to unravel the biological role of hypothetical phage proteins, which constitute an enormous untapped source of novel antibacterial proteins.
Project description:Binding of transcription factors to DNA is mediated by the recognition of the chemical signatures of the DNA bases and the three-dimensional shape of the DNA molecule. The direct contribution of DNA shape to DNA-binding specificity has been difficult to assess, as DNA shape is a consequence of its sequence. Here, we teased apart these two modes of recognition in the context of Hox-DNA binding. We made a series of mutations in Hox residues that, in a co-crystal structure, only recognize DNA shape, and tested the effect on DNA binding preferences using SELEX-seq. Analysis of shape features of selected sequences revealed that these residues are both necessary and sufficient for selection of sequences with distinct shape features. We used statistical machine learning to show that the accuracy of binding specificity predictions improves by adding shape features to a model that only depends on sequence. We conclude that shape readout is a direct and critical component of binding site selection by Hox proteins. Three rounds of SELEX were performed on a series of Hox mutants as described in Slattery et al, Cell, 2011 (PMID 22153072) and Riley et al, Methods in molecular Biology, 2014 (PMID 25151169). Briefly, His-tagged Scr and Antp mutant proteins were incubated with a randomized 16mer oligonucleotide library, and bound DNA was amplified and sequenced as described (PMID 22153072, PMID 25151169).