scholarly journals Crystal structure of an Escherichia coli Hfq Core (residues 2–69)–DNA complex reveals multifunctional nucleic acid binding sites

2020 ◽  
Vol 48 (7) ◽  
pp. 3987-3997 ◽  
Author(s):  
Jillian Orans ◽  
Alexander R Kovach ◽  
Kirsten E Hoff ◽  
Nicola M Horstmann ◽  
Richard G Brennan
Keyword(s):  
Crystal Structure ◽  
Escherichia Coli ◽  
Nucleic Acid ◽  
Dna Interaction ◽  
Dna Duplexes ◽  
Nucleic Acid Binding ◽  
Binding Studies ◽  
Bacterial Gene ◽  
Bacterial Gene Expression ◽  
Microscopy Data

Abstract Hfq regulates bacterial gene expression post-transcriptionally by binding small RNAs and their target mRNAs, facilitating sRNA-mRNA annealing, typically resulting in translation inhibition and RNA turnover. Hfq is also found in the nucleoid and binds double-stranded (ds) DNA with a slight preference for A-tracts. Here, we present the crystal structure of the Escherichia coli Hfq Core bound to a 30 bp DNA, containing three 6 bp A-tracts. Although previously postulated to bind to the ‘distal’ face, three statistically disordered double stranded DNA molecules bind across the proximal face of the Hfq hexamer as parallel, straight rods with B-DNA like conformational properties. One DNA duplex spans the diameter of the hexamer and passes over the uridine-binding proximal-face pore, whereas the remaining DNA duplexes interact with the rims and serve as bridges between adjacent hexamers. Binding is sequence-independent with residues N13, R16, R17 and Q41 interacting exclusively with the DNA backbone. Atomic force microscopy data support the sequence-independent nature of the Hfq-DNA interaction and a role for Hfq in DNA compaction and nucleoid architecture. Our structure and nucleic acid-binding studies also provide insight into the mechanism of sequence-independent binding of Hfq to dsRNA stems, a function that is critical for proper riboregulation.

Metal homeostasis in Escherichia coli: The regulation of bacterial gene expression by cupric and mercuric ions.

1991 ◽  
Vol 43 (2-3) ◽  
pp. 504
Author(s):  
Nigel L. Brown ◽  
Siobhán R. Barrett ◽  
Barry T.O. Lee ◽  
Julian Parkhill ◽  
Duncan A. Rouch
Keyword(s):  
Gene Expression ◽  
Escherichia Coli ◽  
Metal Homeostasis ◽  
Bacterial Gene ◽  
Bacterial Gene Expression ◽  
Mercuric Ions

scholarly journals Flow Cytometric Monitoring of Antibiotic-Induced Injury in Escherichia coli Using Cell-Impermeant Fluorescent Probes

2000 ◽  
Vol 44 (3) ◽  
pp. 676-681 ◽  
Author(s):  
Fiona C. Mortimer ◽  
David J. Mason ◽  
Vanya A. Gant
Keyword(s):  
Escherichia Coli ◽  
Nucleic Acid ◽  
Fluorescence Intensity ◽  
Antimicrobial Agents ◽  
Flow Cytometric Analysis ◽  
Bacterial Cells ◽  
Nucleic Acid Binding ◽  
Permeabilized Cells ◽  
Sytox Green ◽  
Flow Cytometric

ABSTRACT Three fluorescent nucleic acid binding dyes—propidium iodide, TO-PRO-1, and SYTOX green—were evaluated, and their abilities to distinguish between bacterial cells with and without an intact cytoplasmic membrane were compared. Each dye was readily able to discriminate between healthy and permeabilized cells ofEscherichia coli, although SYTOX green showed a greater enhancement in fluorescence intensity on staining-compromised, as opposed to healthy, cells in log-phase growth, than either PI or TO-PRO-1. Flow cytometric analysis of E. coli stained with these dyes after exposing them to several antimicrobial agents showed that all three dyes were able to detect antimicrobial action. Notably, however, the intensity of the cell-associated fluorescence was related to the mechanism of action of the antimicrobial agent. Large changes in fluorescence intensity were observed for all the dyes subsequent to β-lactam antibiotic action, but smaller changes (or no change) were seen subsequent to exposure to antimicrobials acting directly or indirectly on nucleic acid synthesis. Furthermore, cell-associated fluorescence did not relate to loss of viability as determined by plate counts. Despite offering much insight into antimicrobial mechanisms of action, these fundamental problems become relevant to the development of rapid antimicrobial susceptibility tests if colony formation is used as the standard.

scholarly journals Flow Cytometric Investigation of Filamentation, Membrane Patency, and Membrane Potential in Escherichia coli following Ciprofloxacin Exposure

2000 ◽  
Vol 44 (3) ◽  
pp. 682-687 ◽  
Author(s):  
H. J. Wickens ◽  
R. J. Pinney ◽  
D. J. Mason ◽  
V. A. Gant
Keyword(s):  
Escherichia Coli ◽  
Protein Synthesis ◽  
Cell Division ◽  
Membrane Potential ◽  
Nucleic Acid ◽  
Nutrient Broth ◽  
Nucleic Acid Binding ◽  
Dye Uptake ◽  
Flow Cytometric ◽  
Drug Removal

ABSTRACT Ninety-eight percent of the cells in a population ofEscherichia coli in log-phase growth lost colony-forming ability after being exposed for 3 h to the quinolone antibiotic ciprofloxacin at four times the MIC in nutrient broth, a concentration easily reached in vivo. Flow cytometric analysis, however, demonstrated that only 68% of this bacterial population had lost membrane potential, as judged by the membrane potential-sensitive dye bis-(1,3-dibutylbarbituric acid) trimethine oxonol [DiBAC4(3)], and only 30% could no longer exclude the nucleic acid-binding dye propidium iodide (PI), reflecting lost membrane integrity, efflux mechanisms, or both. Subsequent removal of ciprofloxacin and resuspension in nutrient broth resulted in renewed cell division after 2 h, with a calculated postantibiotic effect (PAE) time of 57 min. The proportion of DiBAC- and PI-fluorescent cells in this recovering population remained stable for more than 4 h after antibiotic removal. Eighty percent of cells present at drug removal were filamentous. Their number subsequently decreased with time, and the increase in particle count seen at the end of the PAE resulted from the division of short cells. Exposure to ciprofloxacin in the presence of the protein synthesis inhibitor chloramphenicol increased colony-forming ability to 60% of starting population numbers. In contrast to ciprofloxacin alone, this antibiotic combination resulted in insignificant filamentation and no dye uptake. Subsequent drug removal and resuspension in nutrient broth resulted in the appearance of filaments within 1 h, with 69% of the population forming filaments at 3 h. Dye uptake was also seen, with 20% of the population fluorescing with either dye after 4 h. We were unable to relate dye uptake to the viable count. Cell division resumed 240 min after removal of both drugs, yielding a PAE calculated at 186 min. Inhibition of protein synthesis with chloramphenicol prevented ciprofloxacin-induced changes in bacterial morphology, cell membrane potential, and ability to exclude nucleic acid-binding dye. These changes persisted beyond the end of the classically defined PAE and were not a definite indicator of cell death as defined by loss of colony formation, which related at least in part to filamentation.

scholarly journals Structural basis of nucleic-acid recognition and double-strand unwinding by the essential neuronal protein Pur-alpha

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Janine Weber ◽  
Han Bao ◽  
Christoph Hartlmüller ◽  
Zhiqin Wang ◽  
Almut Windhager ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Nucleic Acid ◽  
Molecular Mechanisms ◽  
Rna Binding ◽  
Point Mutations ◽  
Structural Basis ◽  
Binding Modes ◽  
Nucleic Acid Binding

The neuronal DNA-/RNA-binding protein Pur-alpha is a transcription regulator and core factor for mRNA localization. Pur-alpha-deficient mice die after birth with pleiotropic neuronal defects. Here, we report the crystal structure of the DNA-/RNA-binding domain of Pur-alpha in complex with ssDNA. It reveals base-specific recognition and offers a molecular explanation for the effect of point mutations in the 5q31.3 microdeletion syndrome. Consistent with the crystal structure, biochemical and NMR data indicate that Pur-alpha binds DNA and RNA in the same way, suggesting binding modes for tri- and hexanucleotide-repeat RNAs in two neurodegenerative RNAopathies. Additionally, structure-based in vitro experiments resolved the molecular mechanism of Pur-alpha's unwindase activity. Complementing in vivo analyses in Drosophila demonstrated the importance of a highly conserved phenylalanine for Pur-alpha's unwinding and neuroprotective function. By uncovering the molecular mechanisms of nucleic-acid binding, this study contributes to understanding the cellular role of Pur-alpha and its implications in neurodegenerative diseases.

scholarly journals Correction: Synthetic approaches to nucleopeptides containing all four nucleobases, and nucleic acid-binding studies on a mixed-sequence nucleo-oligolysine

RSC Advances ◽  
2016 ◽  
Vol 6 (82) ◽  
pp. 78486-78486
Author(s):  
Giovanni N. Roviello ◽  
Domenica Musumeci
Keyword(s):  
Nucleic Acid ◽  
Nucleic Acid Binding ◽  
Binding Studies ◽  
Mixed Sequence ◽  
Synthetic Approaches

Correction for ‘Synthetic approaches to nucleopeptides containing all four nucleobases, and nucleic acid-binding studies on a mixed-sequence nucleo-oligolysine’ by Giovanni N. Roviello et al., RSC Adv., 2016, 6, 63578–63585.

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