December 2010
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10 Reads
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December 2010
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10 Reads
December 2010
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85 Reads
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11 Citations
Nucleic Acids Research
Retroviruses require both spliced and unspliced RNAs for replication. Accumulation of Rous Sarcoma virus (RSV) unspliced RNA depends upon the negative regulator of splicing (NRS). Its 5′-part is considered as an ESE binding SR proteins. Its 3′-part contains a decoy 5′-splice site (ss), which inhibits splicing at the bona fide 5′-ss. Only the 3D structure of a small NRS fragment had been experimentally studied. Here, by chemical and enzymatic probing, we determine the 2D structure of the entire RSV NRS. Structural analysis of other avian NRSs and comparison with all sequenced avian NRSs is in favour of a phylogenetic conservation of the NRS 2D structure. By combination of approaches: (i) in vitro and in cellulo splicing assays, (ii) footprinting assays and (iii) purification and analysis of reconstituted RNP complex, we define a small NRS element retaining splicing inhibitory property. We also demonstrate the capability of the SR protein 9G8 to increase NRS activity in vitro and in cellulo. Altogether these data bring new insights on how NRS fine tune splicing activity.
October 2005
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265 Reads
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26 Citations
Molecular Microbiology
A gapA-pgk gene tandem coding the glyceraldehyde 3-phosphate dehydrogenase and 3-phosphoglycerate kinase, is most frequently found in bacteria. However, in Enterobacteriaceae, gapA is replaced by an epd open reading frame (ORF) coding an erythrose-4-phosphate dehydrogenase and an fbaA ORF coding the class II fructose-1,6-bisphosphate aldolase follows pgk. Although epd expression is very low in Escherichia coli, we show that, in the presence of glucose, the 3 epd, pgk and fbaA ORFs are efficiently cotranscribed from promoter epd P0. Conservation of promoter epd P0 is likely due to its important role in modulation of the metabolic flux during glycolysis and gluconeogenesis. As a consequence, we found that the epd translation initiation region and ORF have been adapted in order to limit epd translation and to create an efficient RNase E entry site. We also show that fbaA is cotranscribed with pgk, from promoter epd P0 or an internal pgk P1 promoter of the extended -10 class. The differential expression of pgk and fbaA also depends upon an RNase E segmentation process, leading to individual mRNAs with different stabilities. The secondary structures of the RNA regions containing the RNase E sites were experimentally determined which brings important information on the structural features of RNase E ectopic sites.
July 1999
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22 Reads
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13 Citations
Biotechnology and Bioengineering
At high glucose concentrations, Escherichia coli produces acetate (Crabtree effect). To look for the influence of glucose and/or acetate in the medium on the expression of a recombinant gene in E. coli, the effect of a pulse addition of glucose, on transcription of a cloned E. coli gapA gene and the resulting glyceraldehyde-3P-dehydrogenase activity (GAPDH), was tested during continuous cultivation of E. coli HB101 transformed with the plasmid pBR::EcogapA. Stable continuous cultures were established in a semi-synthetic medium supplemented with 5 g/L of glucose. After the addition of 7 g of glucose within a few seconds, gapA gene expression was strongly and very rapidly induced. As shown by primer-extension analysis, promoter P1, one of the four transcriptional promoters of the gapA gene, was strongly activated, and GAPDH activity increased. However, after rapid glucose consumption, acetate was produced and acetate concentrations above 2 g/L induced stress conditions. This is shown by a strong activation of promoter P2, that is recognized by the stress specific Esigma32 RNA polymerase. During this period, the total cellular RNA content was strongly diminished. Later, when acetate was partially consumed a high level of total RNA was restored, translation was efficient and a regular increase of the GAPDH-specific activity was observed. The transitions between glucose metabolism, acetate production and the end of acetate consumption, were marked by large increases in RNase and protease activities. For comparison, pulse-addition experiments were also performed with serine and alanine. A transient increase of GAPDH production associated with an increase in biomass was also found for serine that can be utilized as an energy source, whereas the addition of alanine, which is only incorporated into newly synthesized proteins, did not increase GAPDH production. The implication of these data for overproduction of recombinant proteins in E. coli is discussed.
June 1999
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23 Reads
Biotechnology and Bioengineering
At high glucose concentrations, Escherichia coli produces acetate (Crabtree effect). To look for the influence of glucose and/or acetate in the medium on the expression of a recombinant gene in E. coli, the effect of a pulse addition of glucose, on transcription of a cloned E. coli gapA gene and the resulting glyceraldehyde-3P-dehydrogenase activity (GAPDH), was tested during continuous cultivation of E. coli HB101 transformed with the plasmid pBR::EcogapA. Stable continuous cultures were established in a semi-synthetic medium supplemented with 5 g/L of glucose. After the addition of 7 g of glucose within a few seconds, gapA gene expression was strongly and very rapidly induced. As shown by primer-extension analysis, promoter P1, one of the four transcriptional promoters of the gapA gene, was strongly activated, and GAPDH activity increased. However, after rapid glucose consumption, acetate was produced and acetate concentrations above 2 g/L induced stress conditions. This is shown by a strong activation of promoter P2, that is recognized by the stress specific Eσ32 RNA polymerase. During this period, the total cellular RNA content was strongly diminished. Later, when acetate was partially consumed a high level of total RNA was restored, translation was efficient and a regular increase of the GAPDH-specific activity was observed. The transitions between glucose metabolism, acetate production and the end of acetate consumption, were marked by large increases in RNase and protease activities. For comparison, pulse-addition experiments were also performed with serine and alanine. A transient increase of GAPDH production associated with an increase in biomass was also found for serine that can be utilized as an energy source, whereas the addition of alanine, which is only incorporated into newly synthesized proteins, did not increase GAPDH production. The implication of these data for overproduction of recombinant proteins in E. coli is discussed. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 63: 712–720, 1999.
January 1999
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169 Reads
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39 Citations
The Escherichia coli gapB gene codes for a protein that is very similar to bacterial glyceraldehyde-3-phosphate dehydrogenases (GAPDH). In most bacteria, the gene for GAPDH is located upstream of the pgk gene encoding 3-phosphoglycerate kinase (PGK). This is the case for gapB. However, this gene is poorly expressed and encodes a protein with an erythrose 4-phosphate dehydrogenase activity (E4PDH). The active GAPDH is encoded by the gapA gene. Since we found that the nucleotide region upstream of the gapB open reading frame is responsible for part of the PGK production, we analyzed gapB promoter activity in vivo by direct measurement of the mRNA levels by reverse transcription. We showed the presence of a unique transcription promoter, gapB P0, with a cyclic AMP (cAMP) receptor protein (CRP)-cAMP binding site centered 70.5 bp upstream of the start site. Interestingly, the gapB P0 promoter activity was strongly enhanced when glucose was used as the carbon source. In these conditions, deletion of the CRP-cAMP binding site had little effect on promoter gapB P0 activity. In contrast, abolition of CRP production or of cAMP biosynthesis (crp or cya mutant strains) strongly reduced promoter gapB P0 activity. This suggests that in the presence of glucose, the CRP-cAMP complex has an indirect effect on promoter gapB P0 activity. We also showed that glucose stimulation of gapB P0 promoter activity depends on the expression of enzyme IIGlc (EIIGlc), encoded by the ptsG gene, and that the gapA P1 promoter is also activated by glucose via the EIIGlc protein. A similar glucose-mediated activation, dependent on the EIIGlc protein, was described by others for the pts operon. Altogether, this shows that when glucose is present in the growth medium expression of the E. coli genes required for its uptake (pts) and its metabolism (gapA and gapB-pgk) are coordinately activated by a mechanism dependent upon the EIIGlc protein.
June 1996
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10 Reads
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2 Citations
Annals of the New York Academy of Sciences
December 1994
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20 Reads
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10 Citations
Current Microbiology
Escherichia coli ATCC 11105 and JM109, transformed with a multicopy plasmid carrying the penicillin G amidase (PGA) gene, were grown at 26 degrees and 37 degrees C, in the presence or the absence of phenylacetic acid (PAA) or of glucose. A method based on primer extension was developed to quantify in vivo levels of PGA mRNAs. A unique transcription start site was found to be used in all the fermentation conditions tested. This site is located 28 nucleotides upstream of the initiation codon. Its utilization is subjected to catabolic repression and is induced by PAA. This site is used at 37 degrees C, but the PGA mRNA level in E. coli ATCC 11105 is lower at 37 degrees C than at 26 degrees C. Induction of the pga gene by PAA was found to be more efficient in the producer strain. Taking into account the amount of PGA mRNA present in the cells at 37 degrees C, one would expect the production of active PGA at this temperature. This is not the case. Thus, at 37 degrees C, expression is blocked at a step after transcription.
November 1994
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13 Reads
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30 Citations
Applied Microbiology and Biotechnology
The Escherichia coli penicillin G amidase (PGA), which is a key enzyme in the production of penicillin G derivatives is generated from a precursor polypeptide by an unusual internal maturation process. We observed the accumulation of the PGA precursor polypeptide in the insoluble material recovered after sonication of recombinant E. coli JM109 cells grown at 26 degrees C. The aggregated nature of the accumulated molecules was demonstrated using detergents and chaotropic agents in solubilization assays. The periplasmic location of the aggregates was shown by trypsin-accessibility experiments performed on the spheroplast fraction. Finally, we showed that addition of sucrose or glycerol in the medium strongly reduces this periplasmic aggregation and as a consequence PGA production is substantially increased. Thus, periplasmic aggregation of the PGA precursor polypeptide limits PGA production by recombinant E. coli and this limitation can be overcome by addition in the medium of a non-metabolizable sugar, such as sucrose, or of glycerol.
January 1993
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27 Reads
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53 Citations
Biotechnology and Bioengineering
Penicillin G amidase (PGA) is a key enzyme for the industrial production of penicillin G derivatives used in therapeutics. Escherichia coli ATCC 11105 is the more commonly used strain for PGA production. To improve enzyme yield, we constructed various recombinant E. coli HB101 and ATCC 11105 strains. For each strain, PGA production was determined for various concentrations of glucose and phenylacetic and (PAA) in the medium. The E. coli strain, G271, was identified as the best performer (800 U NIPAB/L). This strain was obtained as follows: an E. coli ATCC 11105 mutant (E. coli G133) was first selected based on a low negative effect of glucose on PGA production. This mutant was then transformed with a pBR322 derivative containing the PGA gene. Various experiments were made to try to understand the reason for the high productivity of E. coli G271. The host strain, E. coli G133, was found to be mutated in one (or more) gene(s) whose product(s) act(s) in trans on the PGA gene expression. Its growth is not inhibited by high glucose concentration in the medium. Interestingly, whereas glucose still exerts some negative effect on the PGA production by E. coli G133, PGA production by its transformant (E. coli G271) is stimulated by glucose. The reason for this stimulation is discussed. Transformation of E. coli G133 with a pBR322 derivative containing the Hindlll fragment of the PGA gene, showed that the performance of E. coli G271 depends both upon the host strain properties and the plasmid structure. Study of the production by the less efficient E. coli HB101 derivatives brought some light on the mechanism of regulation of the PGA gene.
... To explore MS RNA binding partners during latency, we initiated a proteomic approach based on affinity chromatography purification of RNA-protein complexes (Maenner et al., 2010;Bar et al., 2011) formed upon incubation of in vitro transcribed tat RNA with protein lysate, followed by protein identification by mass spectrometry. An overview of the processes is shown in Figure 1A. ...
December 2010
Nucleic Acids Research
... Although Chao et al. (2002) developed a thermo-inducible lac promoter to obviate the use of IPTG or lactose in large cultures, thermal changes in these cultures also create problems, such as formation of insoluble inclusion bodies and induction of several proteases which decrease the yield of the recombinant proteins (Jobling et al. 1997 ). On the other hand, posttranslational processing of enzymes, such as penicillin acylase, is blocked above 37 @BULLET C (Robas & Brandlant 1994). Galactose and other galactosides, such as butyl-β- D-galactoside and methyl-β-D-galactoside, can bind the lac repressor protein and induce the lac operon (Barkley et al. 1975, Baldauf et al. 1988 ). ...
December 1994
Current Microbiology
... PGA overexpression in E. coli using strong lac or T7 promoter with IPTG induction generally results in the formation of inclusion bodies in the cytoplasm and/or periplasm due to the fast transcription process (Scherrer et al. 1994;Sriubolmas et al. 1997). Arabinose is an effective inducer for PGA production controlled by strong promoter; in addition, it could be used as an extra carbon source for cell growth (Narayanan et al. 2006b;Xu et al. 2006). ...
November 1994
Applied Microbiology and Biotechnology
... Pm4CL1, PmSPS1 and PmSPS2 were cloned into a custom-made, operon-like expression vector pJKW1565. The genes were preceded by a constitutively expressed pGAP promoter of the E. coli glyceraldehyde-3-phosphate dehydrogenase (GAPDH) 79 , and each gene was preceded by a 23-bp ribosome binding site sequence 80 . tSPY was used as a terminator 81 . ...
January 1999
... 11,12 Hence, zones with different acetate concentrations are formed, which is undesirable as acetate concentrations >2 g/L are already toxic for cells. 13 Furthermore, acetate has several negative effects on growth and recombinant protein production as well as induces the cellular stress response, not the least because E. coli adjusts its expression pattern to growth on a secondary carbon source and vice versa. [14][15][16][17] Hence, potentially less productive or less robust subpopulations can occur that reduce overall process efficiency. ...
July 1999
Biotechnology and Bioengineering
... For instance, cspA mRNA triphosphate 5' end is not subject to RppH monophosphorylation, but this mRNA can still be recognized and cleaved by RNase E (Kime et al., 2009). In addition, epd-phk mRNA cleavage by RNase E is also an example of a direct-access pathway (Bardey et al., 2005;Kime et al., 2009). ...
October 2005
Molecular Microbiology
... Ignatova et al. [26] proved that the addition of PAA to the cultivation medium increased the production of PGA in the cell. The inductive effect of PAA in the medium on the PGA production by E. coli ATCC 11105 has been extensively investigated by several authors previously [22,23,31] and found that the addition of PAA into the fermentation medium stimulated the enzyme synthesis. The bulk addition of PAA decreased the production of biomass, and this was attributed due to the inhibition effect of PAA on the growth of B. badius. ...
January 1993
Biotechnology and Bioengineering