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Hochschild, Ann

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Hochschild

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Ann

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Hochschild, Ann
Author's Publications: search.filters.isAuthorOfPublication.73aac1c0-f741-4cf8-a0a8-f9653d8fbcbb

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Now showing 1 - 7 of 7
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    A bacterial three-hybrid assay detects Escherichia coli Hfq–sRNA interactions in vivo
    (Oxford University Press, 2018) Berry, Katherine E; Hochschild, Ann
    Abstract The interaction of RNA molecules with proteins is a critical aspect of gene regulation across all domains of life. Here, we report the development of a bacterial three-hybrid (B3H) assay to genetically detect RNA–protein interactions. The basis for this three-hybrid assay is a transcription-based bacterial two-hybrid assay that has been used widely to detect and dissect protein–protein interactions. In the three-hybrid assay, a DNA-bound protein with a fused RNA-binding moiety (the coat protein of bacteriophage MS2 (MS2CP)) is used to recruit a hybrid RNA upstream of a test promoter. The hybrid RNA consists of a constant region that binds the tethered MS2CP and a variable region. Interaction between the variable region of the hybrid RNA and a target RNA-binding protein that is fused to a subunit of Escherichia coli RNA polymerase (RNAP) stabilizes the binding of RNAP to the test promoter, thereby activating transcription of a reporter gene. We demonstrate that this three-hybrid assay detects interaction between non-coding small RNAs (sRNAs) and the hexameric RNA chaperone Hfq from E. coli and enables the identification of Hfq mutants with sRNA-binding defects. Our findings suggest that this B3H assay will be broadly applicable for the study of RNA–protein interactions.
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    CbtA toxin of Escherichia coli inhibits cell division and cell elongation via direct and independent interactions with FtsZ and MreB
    (Public Library of Science, 2017) Heller, Danielle M.; Tavag, Mrinalini; Hochschild, Ann
    The toxin components of toxin-antitoxin modules, found in bacterial plasmids, phages, and chromosomes, typically target a single macromolecule to interfere with an essential cellular process. An apparent exception is the chromosomally encoded toxin component of the E. coli CbtA/CbeA toxin-antitoxin module, which can inhibit both cell division and cell elongation. A small protein of only 124 amino acids, CbtA, was previously proposed to interact with both FtsZ, a tubulin homolog that is essential for cell division, and MreB, an actin homolog that is essential for cell elongation. However, whether or not the toxic effects of CbtA are due to direct interactions with these predicted targets is not known. Here, we genetically separate the effects of CbtA on cell elongation and cell division, showing that CbtA interacts directly and independently with FtsZ and MreB. Using complementary genetic approaches, we identify the functionally relevant target surfaces on FtsZ and MreB, revealing that in both cases, CbtA binds to surfaces involved in essential cytoskeletal filament architecture. We show further that each interaction contributes independently to CbtA-mediated toxicity and that disruption of both interactions is required to alleviate the observed toxicity. Although several other protein modulators are known to target FtsZ, the CbtA-interacting surface we identify represents a novel inhibitory target. Our findings establish CbtA as a dual function toxin that inhibits both cell division and cell elongation via direct and independent interactions with FtsZ and MreB.
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    Programmable repression and activation of bacterial gene expression using an engineered CRISPR-Cas system
    (Oxford University Press, 2013) Bikard, David; Jiang, Wenyan; Samai, Poulami; Hochschild, Ann; Zhang, Feng; Marraffini, Luciano A.
    The ability to artificially control transcription is essential both to the study of gene function and to the construction of synthetic gene networks with desired properties. Cas9 is an RNA-guided double-stranded DNA nuclease that participates in the CRISPR-Cas immune defense against prokaryotic viruses. We describe the use of a Cas9 nuclease mutant that retains DNA-binding activity and can be engineered as a programmable transcription repressor by preventing the binding of the RNA polymerase (RNAP) to promoter sequences or as a transcription terminator by blocking the running RNAP. In addition, a fusion between the omega subunit of the RNAP and a Cas9 nuclease mutant directed to bind upstream promoter regions can achieve programmable transcription activation. The simple and efficient modulation of gene expression achieved by this technology is a useful asset for the study of gene networks and for the development of synthetic biology and biotechnological applications.
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    Prion propagation can occur in a prokaryote and requires the ClpB chaperone
    (eLife Sciences Publications, Ltd, 2014) Yuan, Andy; Garrity, Sean J; Nako, Entela; Hochschild, Ann
    Prions are self-propagating protein aggregates that are characteristically transmissible. In mammals, the PrP protein can form a prion that causes the fatal transmissible spongiform encephalopathies. Prions have also been uncovered in fungi, where they act as heritable, protein-based genetic elements. We previously showed that the yeast prion protein Sup35 can access the prion conformation in Escherichia coli. Here, we demonstrate that E. coli can propagate the Sup35 prion under conditions that do not permit its de novo formation. Furthermore, we show that propagation requires the disaggregase activity of the ClpB chaperone. Prion propagation in yeast requires Hsp104 (a ClpB ortholog), and prior studies have come to conflicting conclusions about ClpB's ability to participate in this process. Our demonstration of ClpB-dependent prion propagation in E. coli suggests that the cytoplasmic milieu in general and a molecular machine in particular are poised to support protein-based heredity in the bacterial domain of life. DOI: http://dx.doi.org/10.7554/eLife.02949.001
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    The primary σ factor in Escherichia coli can access the transcription elongation complex from solution in vivo
    (eLife Sciences Publications, Ltd, 2015) Goldman, Seth; Nair, Nikhil U; Wells, Christopher D; Nickels, Bryce E; Hochschild, Ann
    The σ subunit of bacterial RNA polymerase (RNAP) confers on the enzyme the ability to initiate promoter-specific transcription. Although σ factors are generally classified as initiation factors, σ can also remain associated with, and modulate the behavior of, RNAP during elongation. Here we establish that the primary σ factor in Escherichia coli, σ70, can function as an elongation factor in vivo by loading directly onto the transcription elongation complex (TEC) in trans. We demonstrate that σ70 can bind in trans to TECs that emanate from either a σ70-dependent promoter or a promoter that is controlled by an alternative σ factor. We further demonstrate that binding of σ70 to the TEC in trans can have a particularly large impact on the dynamics of transcription elongation during stationary phase. Our findings establish a mechanism whereby the primary σ factor can exert direct effects on the composition of the entire transcriptome, not just that portion that is produced under the control of σ70-dependent promoters. DOI: http://dx.doi.org/10.7554/eLife.10514.001
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    Genetic Reporter System for Positioning of Proteins at the Bacterial Pole
    (American Society of Microbiology, 2012) Fixen, Kathryn R.; Janakiraman, Anuradha; Garrity, Sean; Slade, Daniel J.; Gray, Andrew N.; Karahan, Nilay; Hochschild, Ann; Goldberg, Marcia
    Spatial organization within bacteria is fundamental to many cellular processes, although the basic mechanisms underlying localization of proteins to specific sites within bacteria are poorly understood. The study of protein positioning has been limited by a paucity of methods that allow rapid large-scale screening for mutants in which protein positioning is altered. We developed a genetic reporter system for protein localization to the pole within the bacterial cytoplasm that allows saturation screening for mutants in Escherichia coli in which protein localization is altered. Utilizing this system, we identify proteins required for proper positioning of the Shigella autotransporter IcsA. Autotransporters, widely distributed bacterial virulence proteins, are secreted at the bacterial pole. We show that the conserved cell division protein FtsQ is required for localization of IcsA and other autotransporters to the pole. We demonstrate further that this system can be applied to the study of proteins other than autotransporters that display polar positioning within bacterial cells.
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    Including Oneself and Including Others: Who Belongs in My Country?
    (SAGE Publications, 2010) Hochschild, Ann; Lang, Charles
    To be a first class member of a country, must one have citizenship, the same ethnic or racial background, or the same religion, as most citizens? How does high status relate to beliefs about inclusion? We analyze the 2003 ISSP survey on national identity, focusing on ten wealthy, democratic countries. We find a series of mismatches: a strong sense of being included is often coupled with a desire to exclude others. Countries with extreme public views are not always the countries with political controversy over inclusion. Views of citizens or members of the mainstream religion or race often differ from views of outsiders. Countries often cluster in ways that violate standard assumptions about geographic, cultural, or political affinities. Enjoying high status does not guarantee feeling included or seeking to include others. Given these mismatches, it is no surprise that politics and policies around inclusion are contentious, unstable, and fascinating.