Strong nucleic acid binding to the Escherichia coli RNase HI mutant with two arginine residues at the active site

2001 ◽  
Vol 1547 (1) ◽  
pp. 135-142 ◽  
Author(s):  
Yasuo Tsunaka ◽  
Mitsuru Haruki ◽  
Masaaki Morikawa ◽  
Shigenori Kanaya
Keyword(s):  
Escherichia Coli ◽  
Nucleic Acid ◽  
Active Site ◽  
Nucleic Acid Binding ◽  
Arginine Residues ◽  
Rnase Hi

How RNase HI (Escherichia coli) promoted site-selective hydrolysis works on RNA in duplex with carba-LNA and LNA substituted antisense strands in an antisense strategy context?

2017 ◽  
Vol 13 (5) ◽  
pp. 921-938
Author(s):  
Oleksandr Plashkevych ◽  
Qing Li ◽  
Jyoti Chattopadhyaya
Keyword(s):  
Escherichia Coli ◽  
Nucleic Acid ◽  
Kinetic Study ◽  
Rnase H ◽  
Locked Nucleic Acid ◽  
Cleavage Sites ◽  
Selective Hydrolysis ◽  
Rnase Hi ◽  
Site Selective

Kinetic study of 36 AON–RNA heteroduplexes single modified by locked nucleic acid (LNA) or by carba-LNA show site-dependent modulation of RNase H promoted cleavage of RNA strand by 2 to 5 fold with preferential 5′-GpN-3′ cleavage sites, giving up to 70% of the products.

scholarly journals Role of αArg145 and βArg263 in the active site of penicillin acylase of Escherichia coli

2002 ◽  
Vol 365 (1) ◽  
pp. 303-309 ◽  
Author(s):  
Wynand B.L. ALKEMA ◽  
Antoon K. PRINS ◽  
Erik de VRIES ◽  
Dick B. JANSSEN
Keyword(s):  
Escherichia Coli ◽  
Active Site ◽  
Catalytic Mechanism ◽  
Penicillin Acylase ◽  
Precursor Protein ◽  
Site Directed Mutagenesis ◽  
Penicillin G ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
Arginine Residues

The active site of penicillin acylase of Escherichia coli contains two conserved arginine residues. The function of these arginines, αArg145 and βArg263, was studied by site-directed mutagenesis and kinetic analysis of the mutant enzymes. The mutants αArg145→Leu (αArg145Leu), αArg145Cys and αArg145Lys were normally processed and exported to the periplasm, whereas expression of the mutants βArg263Leu, βArg263Asn and βArg263Lys yielded large amounts of precursor protein in the periplasm, indicating that βArg263 is crucial for efficient processing of the enzyme. Either modification of both arginine residues by 2,3-butanedione or replacement by site-directed mutagenesis yielded enzymes with a decreased specificity (kcat/Km) for 2-nitro-5-[(phenylacetyl)amino]benzoic acid, indicating that both residues are important in catalysis. Compared with the wild type, the αArg145 mutants exhibited a 3–6-fold-increased preference for 6-aminopenicillanic acid as the deacylating nucleophile compared with water. Analysis of the steady-state parameters of these mutants for the hydrolysis of penicillin G and phenylacetamide indicated that destabilization of the Michaelis—Menten complex accounts for the improved activity with β-lactam substrates. Analysis of pH—activity profiles of wild-type enzyme and the βArg263Lys mutant showed that βArg263 has to be positively charged for catalysis, but is not involved in substrate binding. The results provide an insight into the catalytic mechanism of penicillin acylase, in which αArg145 is involved in binding of β-lactam substrates and βArg263 is important both for stabilizing the transition state in the reaction and for correct processing of the precursor protein.

scholarly journals SAMHD1 is a single-stranded nucleic acid binding protein with no active site-associated nuclease activity

2015 ◽  
Vol 43 (13) ◽  
pp. 6486-6499 ◽  
Author(s):  
Kyle J. Seamon ◽  
Zhiqiang Sun ◽  
Luda S. Shlyakhtenko ◽  
Yuri L. Lyubchenko ◽  
James T. Stivers
Keyword(s):  
Nucleic Acid ◽  
Active Site ◽  
Binding Protein ◽  
Nuclease Activity ◽  
Nucleic Acid Binding ◽  
Nucleic Acid Binding Protein ◽  
Acid Binding Protein

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 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.

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