Expression of Escherichia coli ribosomal protein and RNA polymerase genes cloned on plasmids

Fiil, Niels P.; Bendiak, Dirk S.; Collins, John T.; Friesen, James D.

doi:10.1007/bf00267689

Cited by 76 publications

(42 citation statements)

References 32 publications

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“…2), cloned in pBR322 (12). The recA mutation prevented marker b Ampicillin (50 pug/ml) was added to all plates to maintain the plasmids.…”

Section: Resultsmentioning

confidence: 99%

“…Plasmid DNA extraction, transformation, and DNA electrophoresis on agarose gels were carried out as described previously (32). Plasmid pNF1492 (12) was kindly provided by J.-P. Bouch6. We constructed plasmid pDV1 from pNF1492 by deletion of a 1.5-kbp Hindlll-Hindlll fragment inactivating the rpUJ gene, coding for protein L10, and we constructed pDV2 from pDV1 by deletion of a 3.2-kbp EcoRV-EcoRV fragment inactivating the rpoB gene (see Fig.…”

Section: Materlals and Methodsmentioning

confidence: 99%

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Thermoinducible filamentation in Escherichia coli due to an altered RNA polymerase beta subunit is suppressed by high levels of ppGpp

Vinella

D’Ari

1994

J Bacteriol

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The Escherichia coli strain known as GC2553, FB8, UTH1038, or K12S (Luria), considered an F-Xwild-type strain, is shown here to carry a cryptic mutation, ftsRl, causing nonlethal filamentation during exponential growth in Luria-Bertani (LB) broth at 42°C and the inability to grow in salt-free LB broth at 42°C.The ftsRl mutation is completely suppressed in genetic backgrounds which increase RelA-dependent synthesis of the nucleotide ppGpp, i.e., argS201(Mecr) and alaS21(Mecr) mutations, affecting aminoacyl-tRNA synthetases, or the presence of a pc-reU' plasmid. These backgrounds also confer resistance in LB broth to the I-lactam mecillinam, an antibiotic which specifically inhibits penicillin-binding protein 2 and, in wild-type cells, causes an indirect block in cell division. Furthermore, the ftsRI mutant (but not an isogenicftsR' strain) is sensitive to mecillinam in minimal glucose medium at 37°C. Since the division block caused by mecillinam can be overcome by overproduction of the cell division protein FtsZ, we tested the effect of plasmid pZAQ (carrying the ftsZ, ftsA, and ftsQ genes) on the ftsRl mutant; it suppressed the filamentation in LB broth and the mecillinam sensitivity on minimal glucose medium at 37°C but not the growth defect in salt-free LB broth at 42°C. Genetic analysis indicated that the full phenotype of the ftsRl mutant is due to a single mutation in the rpoB gene (90 min), coding for the P subunit of RNA polymerase; we call this allele rpoB369(Fts). We propose that the rpoB369(Fts) mutation alters the specificity of the polymerase and that the mutant enzyme can recover normal activity in the presence of high salt concentrations or via interaction with the nucleotide ppGpp.The cell cycle of Escherichia coli is tightly regulated. Septation normally begins when cells reach a precise length, suggesting the existence of a mechanism for coupling between cell wall elongation and septation. Such coupling mechanisms ensure a harmonious cell cycle. To understand them, one must identify the effectors and their targets, presumably elements of the septation apparatus. The isolation of conditional (fts) mutants that cannot divide at nonpermissive temperatures has been extensively used to define elements involved in E. coli cell division. This approach identified the essential gene ftsZ, whose product is required for an early step of septation and seems to be limiting for the process (45,47). This protein is a key control point of septation and the target of several endogenous cell division inhibitors (24). It has been shown that FtsZ, which is normally cytoplasmic, forms a ring around the cell center at the time of septation (3) and that this ring cannot be formed when the cell division inhibitor SfiA or MinCD is overexpressed (4). The FtsZ protein possesses a GTPase activity essential for septation; its GTP-binding site is homologous to those of eukaryotic tubulins, suggesting that the FtsZ protein may have a structural rather than an enzymatic function in septation (11,27,31).The peptidoglycan synthe...

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“…2), cloned in pBR322 (12). The recA mutation prevented marker b Ampicillin (50 pug/ml) was added to all plates to maintain the plasmids.…”

Section: Resultsmentioning

confidence: 99%

Section: Materlals and Methodsmentioning

confidence: 99%

Thermoinducible filamentation in Escherichia coli due to an altered RNA polymerase beta subunit is suppressed by high levels of ppGpp

Vinella

D’Ari

1994

J Bacteriol

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“…2). These plasmids were derived from pNF1931 (8 The 10-kb HindIII fragment containing the mutated gene from strain SPE75 was isolated by cloning in pBR322 by using a Rif' marker to screen for the P-P'-encoding plasmid which is analogous to pNF1931 (Fig. 3).…”

Section: Resultsmentioning

confidence: 99%

A missense mutation in the rpoC gene affects chromosomal replication control in Escherichia coli

Petersen

Hansen

1991

J Bacteriol

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An RNA polymerase mutant with a single-base-pair change in the rpoC gene affects chromosome initiation control. The mutation, which is recessive, is a G to A transition leading to the substitution of aspartate for glycine at amino acid residue 1033 in the RNA polymerase beta' subunit. The chromosome copy number is increased twofold in the mutant at semipermissive growth temperatures (39 degrees C). In a delta oriC strain, in which chromosome initiation is governed by an F replicon, chromosome copy number is not affected. Plasmid pBR322 copy number is also increased in the mutant at 39 degrees C. The mutation causes a more than fivefold increased expression of the dnaA gene at 39 degrees C. It is conceivable that it is this high DnaA concentration which causes the high chromosome copy number and that the mutant RNA polymerase beta' subunit exerts its effect by altering the expression of the dnaA gene. However, other factors must be affected as well to explain why the RNA polymerase mutant can grow in a balanced fashion with a high chromosome concentration. This is in contrast to wild-type cells, which exhibit higher origin concentrations when DnaA protein is overproduced, but in which the overall DNA concentration is only moderately affected.

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“…RL670 was obtained by selecting for double crossover of the rpoC3530 zja::kan region from pRL648 in a pRL648 transformant of JC7623 (33) using 25 g of kanamycin/ml in LB agar plates as described by Oden et al (34). pRL648 was derived from the rpoCcarrying plasmid pRW207 in the following steps: (i) introduction of XhoI and StuI sites at the C terminus of rpoC by oligonucleotide-directed mutagenesis of a single-stranded form of pRW207 to yield pRL622 (oligonucleotide: 5Ј-TACGTTATTTGAGGCCTAACTCGAGCTCGTTA-TCAGAACCGCCCA-3Ј); (ii) ligation of a 288-bp XhoI, StuI-digested PCR fragment containing the C-terminal 86 codons of E. coli fabE from chromosomal DNA of strain CY15001 between the XhoI and StuI sites of pRL622 to yield pRL625 (PCR oligonucleotides: 5Ј-CACCTCGAG-GCGCCAGCAGCAGCGGAAATCA-3Ј and 5Ј-CACAGGCCTCAATTT-TATCCAGCATCTTCG-3Ј); (iii) ligation into the HindIII site of pRL625 of a 1374-bp HindIII-Eco47III fragment carrying kan from the E. coli transposon Tn5 (proximal to rpoC; obtained from the plasmid pKIXX from Amersham Biosciences) and a 2326-bp SspI-HindIII fragment carrying the region immediately downstream of rpoC from pNF1346 (distal to rpoC (35)) to yield pRL648. pRW207 is identical to pRW308 (36) except that it contains a wild-type lacI gene and a wild-type rpoC translation initiation signal.…”

Section: Methodsmentioning

confidence: 99%

Diversity in the Rates of Transcript Elongation by Single RNA Polymerase Molecules

Nørrelykke

Engh

Landick

et al. 2004

Journal of Biological Chemistry

102

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Single-molecule measurements of the activities of a variety of enzymes show that rates of catalysis may vary markedly between different molecules in putatively homogenous enzyme preparations. We measured the rate at which purified Escherichia coli RNA polymerase moves along a ϳ2650-bp DNA during transcript elongation in vitro at 0.5 mM nucleoside triphosphates. Individual molecules of a specifically biotinated RNA polymerase derivative were tagged with 199-nm diameter avidin-coated polystyrene beads; enzyme movement along a surface-linked DNA molecule was monitored by observing changes in bead Brownian motion by light microscopy. The DNA was derived from a naturally occurring transcription unit and was selected for the absence of regulatory sequences that induce lengthy pausing or termination of transcription. With rare exceptions, individual enzyme molecules moved at a constant velocity throughout the transcription reaction; the distribution of velocities across a population of 140 molecules was unimodal and was well fit by a Gaussian. However, the width of the Gaussian, ‫؍‬ 6.7 bp/s, was considerably larger than the precision of the velocity measurement (1 bp/s). The observations show that different transcription complexes have differences in catalytic rate (and thus differences in structure) that persist for thousands of catalytic turnovers. These differences may provide a parsimonious explanation for the complex transcription kinetics observed in bulk solution.Different individual molecules within a purified enzyme or ribozyme preparation can display greatly differing rate constants for catalytic turnover (1-3). Some of this variability may be due to the presence of enzyme molecules with differing covalent structures; these may arise from the presence of two or more related genes, from alternative splicing of a single pre-mRNA, or from post-translational protein modifications (4). However, some heterogeneity may also arise from the adoption by enzyme molecules with identical covalent structures of different conformational states that persist through multiple catalytic turnovers (5, 6). It has been proposed that such conformational heterogeneity may be a heretofore unappreciated feature of macromolecular catalysts in general (2).Bacterial RNA polymerases (RNAPs), 1 and their structurally and mechanistically similar homologs, eukaryotic RNAP II enzymes, are large, multisubunit proteins that synthesize all cellular mRNAs (7,8). These enzymes are processive; a given RNA molecule is synthesized in its entirety by the same RNAP molecule. In bacterial RNAPs, most RNA synthesis takes place in a stable transcription elongation complex (TEC) containing the core RNAP subunits, the template DNA, and the nascent RNA transcript. The TEC structure has been extensively characterized (9 -12). A variety of proteins and DNA sequence elements in both prokaryotes and eukaryotes regulate gene expression by altering the extent to which active TECs terminate transcription upstream of or within genes (13-15). The efficiency of termi...

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Expression of Escherichia coli ribosomal protein and RNA polymerase genes cloned on plasmids

Cited by 76 publications

References 32 publications

Thermoinducible filamentation in Escherichia coli due to an altered RNA polymerase beta subunit is suppressed by high levels of ppGpp

Thermoinducible filamentation in Escherichia coli due to an altered RNA polymerase beta subunit is suppressed by high levels of ppGpp

A missense mutation in the rpoC gene affects chromosomal replication control in Escherichia coli

Diversity in the Rates of Transcript Elongation by Single RNA Polymerase Molecules

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