<i>E.coli</i>host strains significantly affect the quality of small scale plasmid DNA preparations used for sequencing

Taylor, R.; Walker, David C.; McInnes, Roderick R.

doi:10.1093/nar/21.7.1677

Cited by 243 publications

(119 citation statements)

References 8 publications

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“…Strains-The E. coli strains used were DH5␣ (FЈ fhuA2 ⌬(argF-lacZ)U169 phoA glnV44 ⌽80 ⌬(lacZ)M15 gyrA96 recA1 relA1 endA1 thi-1 hsdR17a) (27) for cloning experiments and BL21(DE3) (F Ϫ r B Ϫ m B Ϫ gal ompT (int::P lacUV5 T7 gen1 imm21 nin5) (28) for expression and purification of enzymes.…”

Section: Methodsmentioning

confidence: 99%

Determination of Key Residues for Catalysis and RNA Cleavage Specificity

Barbas

Matos

Amblar

et al. 2009

Journal of Biological Chemistry

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RNase II is the prototype of a ubiquitous family of enzymes that are crucial for RNA metabolism. In Escherichia coli this protein is a single-stranded-specific 3-exoribonuclease with a modular organization of four functional domains. In eukaryotes, the RNase II homologue Rrp44 (also known as Dis3) is the catalytic subunit of the exosome, an exoribonuclease complex essential for RNA processing and decay. In this work we have performed a functional characterization of several highly conserved residues located in the RNase II catalytic domain to address their precise role in the RNase II activity. We have constructed a number of RNase II mutants and compared their activity and RNA binding to the wild type using different singleor double-stranded substrates. The results presented in this study substantially improve the RNase II model for RNA degradation. We have identified the residues that are responsible for the discrimination of cleavage of RNA versus DNA. We also show that the Arg-500 residue present in the RNase II active site is crucial for activity but not for RNA binding. The most prominent finding presented is the extraordinary catalysis observed in the E542A mutant that turns RNase II into a "super-enzyme."Exoribonuclease II (RNase II) 5 is one of the major exoribonucleases involved in the degradation of RNA (1). This protein degrades RNA hydrolytically in the 3Ј to 5Ј direction, releasing 5Ј-nucleotide monophosphates in a processive manner. Its activity is sequence independent but sensitive to secondary structures, and poly(A) is the preferential substrate for this enzyme (2-6). In Escherichia coli RNase II is responsible for 90% of the hydrolytic activity in crude extracts (7). Moreover, its expression is differentially regulated at the transcriptional and post-transcriptional levels (8 -10), and the protein can be regulated by environmental conditions (10). The E. coli enzyme is the prototype of a widespread family of exoribonucleases, and RNase II homologues can be found in prokaryotes and eukaryotes (11). In the nucleus and in the cytoplasm of eukaryotic cells, RNase II homologues (Rrp44/Dis3) are part of the exosome, an essential multiprotein complex of exoribonucleases involved in processing, turnover, and quality control of different types of RNAs (12). Rrp44 is the only catalytically active nuclease in the human and yeast core exosome (13,14). Moreover, it was recently demonstrated that apart from the RNB catalytic domain, Rrp44p has a second nuclease domain, the PINc domain, that confers endonucleolytic activity to the protein (15, 16). In prokaryotic cells, apart from RNase II, there is an additional RNase II-like enzyme, RNase R, that is involved in RNA degradation and RNA and protein quality control (17)(18)(19)(20). RNase R has also been shown to be required for virulence (21).The determination of E. coli RNase II structure (22-24) showed that, contrary to the sequence predictions, RNase II consisted of four domains; they are two N-terminal cold shock domains (CSD1 and CSD2) involved in RNA b...

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Section: Methodsmentioning

confidence: 99%

Determination of Key Residues for Catalysis and RNA Cleavage Specificity

Barbas

Matos

Amblar

et al. 2009

Journal of Biological Chemistry

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“…Bacterial Strains and Growth Conditions-E. coli JM109 or DH5␣ (11,12) was used as a carrier for the plasmids described. All plasmids used in this study are listed in supplemental Table 1.…”

Section: Methodsmentioning

confidence: 99%

New Insights into the Signaling Mechanism of the pH-responsive, Membrane-integrated Transcriptional Activator CadC of Escherichia coli

Haneburger

Eichinger

Skerra

et al. 2011

Journal of Biological Chemistry

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The membrane-integrated transcriptional regulator CadC of Escherichia coli activates expression of the cadBA operon at low external pH with concomitantly available lysine, providing adaptation to mild acidic stress. CadC is a representative of the ToxR-like proteins that combine sensory, signal transduction, and DNA-binding activities within a single polypeptide. Although several ToxR-like regulators such as CadC, as well as the main regulator of Vibrio cholerae virulence, ToxR itself, which activate gene expression at acidic pH, have been intensively investigated, their molecular activation mechanism is still unclear. In this study, a structure-guided mutational analysis was performed to elucidate the mechanism by which CadC detects acidification of the external milieu. Thus, a cluster of negatively charged amino acids (Asp-198, Asp-200, Glu-461, Glu-468, and Asp-471) was found to be crucial for pH detection. These amino acids form a negatively charged patch on the surface of the periplasmic domain of CadC that stretches across its two subdomains. The results of different combinations of amino acid replacements within this patch indicated that the N-terminal subdomain integrates and transduces the signals coming from both subdomains to the transmembrane domain. Alterations in the phospholipid composition did not influence pH-dependent cadBA expression, and therefore, interplay of the acidic surface patch with the negatively charged headgroups is unlikely. Models are discussed according to which protonation of these acidic amino acid side chains reduces repulsive forces between the two subdomains and/or between two monomers within a CadC dimer and thereby enables receptor activation upon lowering of the environmental pH.

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“…17 This enzyme is not essential and its biological role in E. coli is still unknown as mutants resemble wild-type cells in terms of growth rate, phage propagation and conjugation properties. 18 Various studies 19,20 have found that EndA − strains produce DNA of higher quality than EndA + E. coli strains. A DNA fragment containing endA 21 was used to construct an M13 Km R suicide vector.…”

Section: Correspondence: J Crouzetmentioning

confidence: 99%

pCOR: a new design of plasmid vectors for nonviral gene therapy

et al. 1999

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A totally redesigned host/vector system with improved initiator protein, protein, encoded by the pir gene limiting properties in terms of safety has been developed. The its host range to bacterial strains that produce this transpCOR plasmids are narrow-host range plasmid vectors for acting protein; (2) the plasmid's selectable marker is not an nonviral gene therapy. These plasmids contain a conantibiotic resistance gene but a gene encoding a bacterial ditional origin of replication and must be propagated in a suppressor tRNA. Optimized E. coli hosts supporting specifically engineered E. coli host strain, greatly reducing pCOR replication and selection were constructed. High the potential for propagation in the environment or in yields of supercoiled pCOR monomers were obtained (100 treated patients. The pCOR backbone has several features mg/l) through fed-batch fermentation. pCOR vectors carrythat increase safety in terms of dissemination and selecing the luciferase reporter gene gave high levels of lucifertion: (1) the origin of replication requires a plasmid-specific ase activity when injected into murine skeletal muscle.

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E.colihost strains significantly affect the quality of small scale plasmid DNA preparations used for sequencing

Cited by 243 publications

References 8 publications

Determination of Key Residues for Catalysis and RNA Cleavage Specificity

Determination of Key Residues for Catalysis and RNA Cleavage Specificity

New Insights into the Signaling Mechanism of the pH-responsive, Membrane-integrated Transcriptional Activator CadC of Escherichia coli

pCOR: a new design of plasmid vectors for nonviral gene therapy

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