A new vector-host system for construction of lacZ transcriptional fusions where only low-level gene expression is desirable

Podkovyrov, S M; Larson, Timothy J.

doi:10.1016/0378-1119(95)00053-9

Cited by 28 publications

(29 citation statements)

References 10 publications

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“…We used PCR amplification to generate DNA fragments containing different regions upstream of rraA extending up to, and including, the menA promoter, as shown in Fig. 3A, and cloned each fragment upstream of the lacZ gene in a multicopy transcriptional fusion vector, pSP417 (29). In this way, we generated plasmids pMZ002 ([nt Ϫ1076 to Ϫ1]-lacZ), pMZ003 ([nt Ϫ92 to Ϫ1]-lacZ), and pMZ004 ([nt Ϫ1076 to Ϫ93]-lacZ); pMZ001 was the negative-control vector.…”

Section: Resultsmentioning

confidence: 99%

“…We used PCR amplification to generate DNA fragments containing different regions upstream of rraA, i.e., nt Ϫ1076 to Ϫ1 of the rraA upstream region, which includes the PmenA, menA coding sequence, and menA-rraA intergenic region; nt Ϫ1076 to Ϫ93, which includes the PmenA and menA coding sequence; and nt Ϫ92 to Ϫ1 of the menA-rraA intergenic region, and cloned each fragment upstream of the lacZ gene in a multicopy transcriptional fusion vector, pSP417 (29). So we generated plasmids pMZ002 ([nt Ϫ1076 to Ϫ1]-lacZ), pMZ003 ([nt Ϫ92 to Ϫ1]-lacZ), and pMZ004 ([nt Ϫ1076 to Ϫ93]-lacZ); pMZ001 was the negative-control vector.…”

Section: Methodsmentioning

confidence: 99%

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Regulation of RraA, a Protein Inhibitor of RNase E-Mediated RNA Decay

et al. 2006

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Cell 114:623-634, 2003). We show that rraA is expressed from its own promoter, P rraA , located in the menA-rraA intergenic region. Primer extension and lacZ fusion analysis revealed that transcription from P rraA is elevated upon entry into stationary phase in a s -dependent manner. In addition, the stability of the rraA transcript is dependent on RNase E activity, suggesting the involvement of a feedback circuit in the regulation of the RraA level in E. coli.RNase E is an essential protein that plays a crucial role in global mRNA metabolism as well as in the maturation of functional RNAs such as rRNAs, tRNAs, tmRNA, and small regulatory RNAs (3,9,18,19,21,28). To date, RNase E homologs have been found in more than 50 eubacteria, archaebacteria, and plants (16). The cellular level and activity of RNase E are subjected to multiple environmental controls. At one level, RNase E synthesis is autoregulated by modulating the half-life of its own mRNA (12,26). In addition, recent studies have revealed that 5Ј-monophosphorylated RNA serves as an allosteric activator of the endonuclease activity (13). Furthermore, the degradation of target RNAs by RNase E is found to be affected globally by endoribonuclease-binding proteins that control the decay and abundance of individual bacterial mRNAs in trans (8,17).RraA (regulator of ribonuclease activity A), is an evolutionarily conserved 17.4-kDa protein with close homologs (Ͼ40% amino acid identity) in bacteria, archaea, proteobacteria, and plants. RraA binds to RNase E with an equilibrium dissociation constant (K D ) in the low-micromolar range and serves as a trans-acting modulator of the endonuclease activity of the enzyme (17). High-affinity binding requires the C-terminal half region of RNase E, which acts as a scaffold for the assembly of a large multiprotein complex called the degradosome (17,36). RraA appears to interact only with the enzyme and not with RNA substrates (17). Gene chip analysis revealed that the action of RraA results in a dramatic change in the global abundance of mRNAs in Escherichia coli, affecting over 15% of all cellular transcripts. Importantly, the gene expression profile that is obtained upon overexpression of RraA is distinct from that obtained upon depletion of RNase E or through the action of RraB, a second trans-acting RNase E inhibitor of E. coli (8).The rraA gene is located downstream of menA, which encodes a 1,4-dihydroxy-2-naphthoic acid octaprenyltransferase that catalyzes the prenylation of the redox mediator menaquinone (32). Transcription of menA appears to occur from a 70 -dependent promoter. Earlier, Meganathan proposed that rraA (formerly designated menG) is transcribed from the menA promoter in a dicistronic mRNA (22). In this study we demonstrate that rraA is transcribed predominantly from its own promoter (P rraA ) located in the intergenic region between the menA and rraA genes. Transcription from P rraA is s dependent and is induced upon entry into stationary phase. Furthermore, we show that the synthesis of RraA is regulated...

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Regulation of RraA, a Protein Inhibitor of RNase E-Mediated RNA Decay

et al. 2006

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“…New England Nuclear Corp. supplied [␥- 32 P]ATP and ␣-35 S-dATP. The glp repressor was purified to homogeneity as previously described (22).…”

Section: Methodsmentioning

confidence: 99%

Action at a distance for negative control of transcription of the glpD gene encoding sn-glycerol 3-phosphate dehydrogenase of Escherichia coli K-12

Yang

Larson

1996

J Bacteriol

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sn-Glycerol 3-phosphate (glycerol-P) is a ubiquitous compound that can be used by Escherichia coli and other organisms. Glycerol-P is a direct precursor for phospholipid biosynthesis (33). Glycerol-P and its precursors, when present in excess, are used as carbon and energy sources. The reactions in the catabolic pathways are catalyzed by the proteins encoded by the genes of the glp regulon (23, 24). To maintain a cellular concentration of glycerol-P optimal for phospholipid biosynthesis, organisms must coordinate the levels of the catabolic activities with the levels of the phospholipid biosynthetic activities. Specific negative regulation of the glp regulon is exerted by the glp repressor (GlpR) (22-24). The affinity of GlpR for each glp operator site is reduced upon the binding of glycerol-P, the inducer for the glp regulon (23, 24).The glp genes have been mapped to three different regions on the E. coli chromosome and are arranged in five different operons (24). The glpFKX operon is located near 89 min on the linkage map of E. coli (4, 42, 45). The glpF gene encodes the glycerol diffusion facilitator, a cytoplasmic membrane protein, and the glpK gene encodes the cytoplasmic glycerol kinase. The function of the protein encoded by glpX is unknown. The glpTQ operon and divergently transcribed glpACB operon are located near 51 min (4, 9-11, 21, 24, 44). The glpT gene encodes the cytoplasmic membrane-associated glycerol-P permease required for active transport of glycerol-P into the cytoplasm, and the glpQ gene encodes the periplasmic glycerophosphodiesterase that catalyzes the hydrolysis of glycerophosphodiesters to glycerol-P and an alcohol. The glpACB operon encodes the three subunits of anaerobic glycerol-P dehydrogenase. Under anaerobic growth conditions, this enzyme is essential for dissimilation of glycerol-P.The glpD gene and divergently transcribed glpEGR operon are located near 77 min (4, 24). The glpEGR genes encode the specific repressor for the glp regulon and two other proteins (GlpE and GlpG) with unknown functions (36,38,49). The glpD gene encodes the aerobic glycerol-P dehydrogenase associated with the cytoplasmic membrane (3, 37). Under aerobic conditions, this dehydrogenase catalyzes the oxidation of glycerol-P to dihydroxyacetone phosphate with the concurrent formation of reduced flavin adenine dinucleotide used in the respiratory chain. Anaerobic repression of glpD expression is mediated by the two-component regulatory system encoded by arcA and arcB (19).Lin and coworkers found that the glpD operon was controlled more tightly by GlpR than was either the glpTQ or glpFKX operon (30-, 26.5-, and 3.2-fold, respectively [12]). This conclusion was reached after measuring the activities of enzymes encoded by these genes (glycerol-P dehydrogenase, glycerol-P permease, and glycerol kinase) in extracts of a strain harboring a thermolabile glp repressor after growth at various temperatures (12). In contrast, it was recently found that the glpFKX operon was more sensitive than the glpD operon to represso...

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“…Promoter-probe vector pSP417 (Podkovyrov and Larson, 1995) is a derivative of pRS415 (Simons et al, 1987) that contains a promoterless lacZ gene downstream of the multiple cloning site and four transcriptional terminators upstream of the multiple cloning site. Cloning of a 302 bp EcoRI-XbaI fragment containing the 268 bp HaeIII-Nru I fragment with the glpAT control region (Fig.…”

Section: Construction Of Recombinant Plasmidsmentioning

confidence: 99%

Action at a distance for glp repressor control of glpTQ transcription in Escherichia coli K‐12

Yang

Gerhardt

Larson

1997

Molecular Microbiology

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SummaryThe adjacent, divergently transcribed glpACB and glpTQ operons of Escherichia coli encode the anaerobic glycerol 3-phosphate dehydrogenase and glycerol 3-phosphate transporter/phosphodiesterase, respectively. These operons are negatively controlled by glp repressor binding to operators that overlap the glpA promoter elements. Using DNase I footprinting, three additional operators (O T 1-3) were identified at positions þ307 to þ359 within the glpT coding region. To assess a potential regulatory role for these remote operators in vivo, a glpT-lacZ transcriptional fusion containing all of the glpA and glpT operators was constructed. The response of this fusion to the glp repressor was compared to fusion constructs in which O T 1 and O T 3 were inactivated, either by deletion or by sitedirected mutagenesis. It was found that repression of glpT conferred by binding of glp repressor to glpA operators was increased about three-to fourfold upon introduction of the remote glpT operators. In addition, two integration host factor (IHF) binding sites were identified downstream of the glpT transcriptional start site at positions þ15 to þ51 and þ193 to þ227. A regulatory role for IHF was demonstrated by showing that repression of glpT mediated by GlpR was decreased about twofold in strains deficient in IHF and that mutations in IHF1 and/or IHF2 decreased repression about two-to threefold. The effect of IHF was apparent only when the remote operators were present. All of the results are consistent with a model of repression involving GlpR binding simultaneously to the glpA and remote glpT operators, with intervening DNA forming a loop.

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A new vector-host system for construction of lacZ transcriptional fusions where only low-level gene expression is desirable

Cited by 28 publications

References 10 publications

Regulation of RraA, a Protein Inhibitor of RNase E-Mediated RNA Decay

Regulation of RraA, a Protein Inhibitor of RNase E-Mediated RNA Decay

Action at a distance for negative control of transcription of the glpD gene encoding sn-glycerol 3-phosphate dehydrogenase of Escherichia coli K-12

Action at a distance for glp repressor control of glpTQ transcription in Escherichia coli K‐12

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