Use of different tRNASer isoacceptor species in vitro to discriminate between the expression of plasmid genes.

Cenatiempo, Y.; Robakis, Nikolaos K.; Meza-Basso, Luís; Brot, Nathan; Weissbach, Herbert; Reid, Brian R.

doi:10.1073/pnas.79.5.1466

Cited by 15 publications

(14 citation statements)

References 24 publications

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“…A limited in vitro translation reaction in which only the first two tRNAs are present was performed, using malEA2-8 and malEA2-8supl mRNAs as substrates. The rate of synthesis of the 2-amino-acid initiation peptide fMetIle was then determined as previously described (9,24,30) (Table 2). Results from this analysis revealed that the rate of translation of the initiation peptide from malEA2-8supl mRNA was approximately 18 times higher than from malEA2-8 mRNA.…”

Section: Resultsmentioning

confidence: 99%

“…In vitro transcription of the malE gene was carried out as described previously (32). In vitro translation and quantitation of the rate of synthesis of the 2-amino-acid initiation peptide Met-Ile from malEA2-8 and malEA2-8supl mRNAs was performed essentially as described by Weissbach and coworkers (9,24,30). The isolation of 70S initiation complexes was carried out as described previously (29).…”

Section: Methodsmentioning

confidence: 99%

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Alterations in the hydrophilic segment of the maltose-binding protein (MBP) signal peptide that affect either export or translation of MBP

1992

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Mutations that reduce the net positive charge within the hydrophilic segments of the signal peptides of several prokaryotic exported proteins can result in a reduction in the rate of protein export, as well as a reduction in protein synthesis (M. N. Hall, J. Gabay, and M. Shwartz, EMBO J. 2:15-19, 1983 revealed that the role of the hydrophilic segment in the export process can be functionally separated from any role in translation. Taken together, these results strongly suggest that the hydrophilic segment of the MBP signal peptide is not involved in a mechanism that couples MBP translation to export and argue against the presence of a mechanism that obligatorily couples translation to protein export in Escherichia coli.Proteins destined for export in Escherichia coli are synthesized as precursor species with an amino-terminal extension termed the signal peptide that is responsible for initiating the export of proteins from the cytoplasm. The periplasmic maltose-binding protein (MBP) of E. coli, the product of the malE gene, is synthesized with a typical bacterial signal peptide that shares little primary amino acid homology with other signal peptides but does exhibit conserved features characteristic of signal peptides of both prokaryotic and eukaryotic origin. The first eight residues of the MBP signal peptide constitute the hydrophilic segment, a distinct region that carries a net positive charge due to the presence of three basic residues. The hydrophilic segment precedes the hydrophobic core, a region devoid of charged residues that is predicted to assume an a-helical conformation. Following the hydrophobic core is the cleavage site for signal peptidase I. (For a comprehensive review of the MBP signal peptide and MBP export, see reference 4.)A specific role for the basic residues of the hydrophilic segment in protein export was first suggested in the loop model (18). The basic residues were proposed to initiate an ionic interaction between the signal peptide and the nega-* Corresponding author.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Alterations in the hydrophilic segment of the maltose-binding protein (MBP) signal peptide that affect either export or translation of MBP

1992

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“…An identical procedure was followed for constructing the recombinant plasmids pBC303.480 and pBC303.481, except that gene fusion was achieved in a different translational phasing. translation coupled system (6). In this system, plasmiddirected protein synthesis is limited to the formation of the N-terminal dipeptide of the gene product instead of the completed protein.…”

Section: Methodsmentioning

confidence: 99%

Nucleotide sequence and expression of the aceK gene coding for isocitrate dehydrogenase kinase/phosphatase in Escherichia coli

et al. 1988

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The flow of isocitrate through the glyoxylate bypass in Escherichia coli is regulated via the phosphorylationdephosphorylation of isocitrate dehydrogenase mediated by a bifunctional enzyme: isocitrate dehydrogenase kinase/phosphatase. The aceK gene coding for this enzyme is part of the polycistronic ace operon, which also includes the aceB and aceA genes coding, respectively, for malate synthase and isocitrate lyase, the two glyoxylate bypass enzymes. The complete nucleotide sequence of a 2,214-base-pair DNA fragment containing the aceK gene and its 5' flanking region has been determined. In vivo experiments based on gene expression in a miniceil system and protein fusion with j-galactosidase, as well as in vitro assays with a plasmid-directed transcription-translation coupled system, have shown that the aceK gene extends over 1,731 nucleotides encoding a 66,528-dalton protein. The 5' flanking region presents an unusual intercistronic structural pattern consisting of two consecutive long dyad symmetries, almost identical in sequence, which can yield very stable stem-loop units. These structures are probably responsible for the drastic downshifting in expression observed in acetate-grown bacteria between the aceK gene and the aceA gene located immediately upstream in the ace operon.Growth of Escherichia coli on acetate as the sole source of carbon and energy requires operation of the anaplerotic sequence known as the glyoxylate bypass (16). In this pathway two specific enzymes, isocitrate lyase and malate synthase, are activated by bacteria to divert isocitrate from the Krebs cycle and prevent the quantitative loss of the acetate carbons as carbon dioxide (3,35,36). The flow of isocitrate through the glyoxylate cycle is regulated via the phosphorylation-dephosphorylation of isocitrate dehydrogenase, the Krebs cycle enzyme which competes for a common substrate with isocitrate lyase (12, 13). When bacteria are grown on glucose, isocitrate dehydrogenase is fully active and unphosphorylated. Conversely, when cells are cultured on acetate, the activity of the enzyme declines drastically, concomitant with its phosphorylation at multiple sites (9, 28). Such a reversible phosphorylation reaction is mediated by a bifunctional enzyme, isocitrate dehydrogenase kinase/phosphatase, which contains both modifying and demodifying activities on the same polypeptide (19,27).The genes coding for isocitrate lyase (aceA) and malate synthase (aceB) are in the same ace operon located at 90 min on the E. coli K-12 linkage map (2, 5), with aceA downstream from aceB (14). The expression of the ace operon is under the transcriptional control of two genes: the iclR gene, which is adjacent to the ace operon, and the fadR gene, which maps at 25 min and is also involved in the regulation of the fatty acid degradation (fad) regulon (5, 32). Recently, it has been demonstrated that the kinase and phosphatase activities which regulate isocitrate dehydrogenase are encoded by a single gene (aceK), which has been cloned and shown to be essential for ...

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“…Our laboratory has recently developed a procedure for investigation of prokaryotic gene expression in an in vitro DNA-directed system by measuring the synthesis of the first di-or tripeptide characteristic of the gene product (1)(2)(3)(4)(5). For example, using the recombinant plasmid pNF1337, which contains the genes for Escherichia coli ribosomal proteins L10 and L12 and the (3 subunit of RNA polymerase, we studied the expression of these three genes in a coupled in vitro system by measuring the formation of fMet-Ala, fMet-Ser, and fMet-Val, respectively (1,2,5).…”

mentioning

confidence: 99%

“…For example, using the recombinant plasmid pNF1337, which contains the genes for Escherichia coli ribosomal proteins L10 and L12 and the (3 subunit of RNA polymerase, we studied the expression of these three genes in a coupled in vitro system by measuring the formation of fMet-Ala, fMet-Ser, and fMet-Val, respectively (1,2,5). The advantage of this system was that a specific di-or tripeptide product could be measured sensitively by thinlayer chromatography (1,2) or by a rapid extraction procedure (4,5). In addition, the system only contained five purified protein factors and also could be used to quantitate the amount of active mRNA present (3).…”

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confidence: 99%

Simplified in vitro system for study of eukaryotic mRNA translation by measuring di- and tripeptide formation.

Cenatiempo¹,

Twardowski²,

Redfield³

et al. 1983

Proc. Natl. Acad. Sci. U.S.A.

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An in vitro system for measurement of rabbit globin mRNA translation has been developed based on the formation of the NH2-terminal dipeptide, fMet-Val. The basic components include a partially purified initiation factor preparation from rabbit reticulocytes supplemented with eukaryotic initiation factor 4A, purified and formylated yeast Met-tRNA;, and rabbit liver or Escherichia coh Val-tRNA vj1. Picomole quantities of fMetVal are synthesized, dependent on mRNA, and the dipeptide is readily assayed by a simple extraction procedure. In the presence of Leu-tRNA or His-tRNA, the tripeptides fMet-Val-Leu and fMetVal-His are synthesized, corresponding to the NH2-terminal sequence of a-and 3globin, respectively. Therefore, tripeptide synthesis provides a simple means to distinguish between the expression of the a-and P-globin mRNA species.Our laboratory has recently developed a procedure for investigation of prokaryotic gene expression in an in vitro DNA-directed system by measuring the synthesis of the first di-or tripeptide characteristic of the gene product (1-5). For example, using the recombinant plasmid pNF1337, which contains the genes for Escherichia coli ribosomal proteins L10 and L12 and the (3 subunit of RNA polymerase, we studied the expression of these three genes in a coupled in vitro system by measuring the formation of fMet-Ala, fMet-Ser, and fMet-Val, respectively (1, 2, 5). The advantage of this system was that a specific di-or tripeptide product could be measured sensitively by thinlayer chromatography (1,2) or by a rapid extraction procedure (4, 5). In addition, the system only contained five purified protein factors and also could be used to quantitate the amount of active mRNA present (3). Finally, because di-or tripeptide formation indicated that proper initiation of both transcription and translation occurred, the system was ideally suited for studies on the regulation of gene expression.Crystal et aL (6)(7)(8) initially attempted to measure in vitro translation of globin mRNA in eukaryotic systems by assaying for di-and tripeptide synthesis. They did demonstrate that short NH2-terminal peptides were formed in vitro but the mRNA-dependent activity was at the borderline of detection (approximately 50 fmol) and the assay was cumbersome. With the goal of obtaining a simplified in vitro system to study eukaryotic gene expression, we have developed a highly active system for the initiation of globin mRNA translation based on the formation of the first dipeptide of the gene product. In addition, by modifying the system to obtain tripeptide synthesis, it is possible to distinguish between the expression of the a-and ,Bglobin mRNA species. Methods. All procedures were carried out at 40C unless otherwise specified.Preparation of A. salina ribosomes. Unwashed 80S ribosomes were prepared from A. salina dormant embryos according to Zasloff and Ochoa (9). The ribosomal pellets were rinsed with 50 mM Tris HCI, pH 7.4/5 mM KCI/10 mM Mg acetate/ 1 mM dithiothreitol (10), resuspended in 50 mM Tris-HCI,...

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Use of different tRNASer isoacceptor species in vitro to discriminate between the expression of plasmid genes.

Cited by 15 publications

References 24 publications

Alterations in the hydrophilic segment of the maltose-binding protein (MBP) signal peptide that affect either export or translation of MBP

Alterations in the hydrophilic segment of the maltose-binding protein (MBP) signal peptide that affect either export or translation of MBP

Nucleotide sequence and expression of the aceK gene coding for isocitrate dehydrogenase kinase/phosphatase in Escherichia coli

Simplified in vitro system for study of eukaryotic mRNA translation by measuring di- and tripeptide formation.

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