Enhancement of translational efficiency by the Escherichia coli atpE translational initiation region: its fusion with two human genes

McCarthy, John E.G.; Sebald, Walter; Groß, Gerhard; Lammers, Reiner

doi:10.1016/0378-1119(86)90099-5

Cited by 49 publications

(9 citation statements)

References 20 publications

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“…In one case the start codon was also changed (from ATG to GTG; a 242, Table 1). pJLf201 bears the major PL and pn bacteriophage \ promoters in tandem (McCarthy et al. 1986).…”

Section: Cloning Of the Atp Genes And Manipulation Of Their Tir Sequementioning

confidence: 99%

Determinants of translational initiation efficiency in the atp operon of Escherichia coli

McCarthy¹,

Bokelmann²

1988

Molecular Microbiology

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Transcription and translation of the atp genes encoding the subunits b, delta, alpha, gamma and epsilon of the Escherichia coli H+-ATPase were studied. The nature and quantities of the respective transcripts initiated from different promoters were compared with overall expression rates thus yielding accurate information about relative translational efficiency and its coupling to mRNA levels. Part of the highly efficient subunit c gene translational initiation region (TIR) was used as a tool in manipulating the TIRs of the other genes. Rate control of atp cistron translation occurs at the initiation level and is determined locally by each gene's TIR. In this way, individual subunit synthesis rates are set to match the requirements for H+-ATPase assembly. There is no (or very restricted) translational coupling between the cistrons. Translational initiation rates of the normally weakly expressed atp genes could be increased by up to a factor of 27 by manipulating the sequences upstream of the start codons, despite biased codon usages. In the presence of an improved upstream sequence, the N-terminal sequence of the subunit gamma gene exerted a limiting effect. This could be relieved by altering the sequence of the first seven codons. The levels of subunit gamma mRNA were more sensitive to changes in translational efficiency than the concentrations of the other atp mRNAs. The relationships between initiation efficiency and primary and secondary structure in the natural and manipulated atp TIRs are discussed in detail.

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“…In one case the start codon was also changed (from ATG to GTG; a 242, Table 1). pJLf201 bears the major PL and pn bacteriophage \ promoters in tandem (McCarthy et al. 1986).…”

Section: Cloning Of the Atp Genes And Manipulation Of Their Tir Sequementioning

confidence: 99%

Determinants of translational initiation efficiency in the atp operon of Escherichia coli

McCarthy¹,

Bokelmann²

1988

Molecular Microbiology

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“…The exact mechanism of control over the apparent differences in expression exhibited by these genes is currently unknown. McCarthy and coworkers, however, have demonstrated that an RNA sequence within the translational initiation region of uncE causes increased synthesis of the c subunit (15) and can also increase the expression of other genes when located within the translation initiation region of those genes (16). This sequence, therefore, appears to enhance expression of uncE.…”

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confidence: 99%

Use of lacZ fusions to measure in vivo expression of the first three genes of the Escherichia coli unc operon

1989

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We have constructed in-frame lacZ protein fusions to the first three genes of the Escherichia coli unc operon, which codes for the subunits of the proton-translocating ATPase. We have used these constructions to measure the relative in vivo expression of these genes. The second and third genes, uncB and uncE, which code for the a and c subunits of the Fo sector, were expressed at relative levels of approximately 1:10, although the measured expression of uncB depended upon how much of the gene was fused to lacZ. These rates compared favorably with the relative numbers of a and c subunits (a1:cjO) in the purified F1Fo complex. The in vivo expression of uncI, the first gene of the operon, was very low, at best 10 to 20 times less than the expression of uncB. (10,21,22).The genes which code for the eight subunits of the ATPase are contained within the unc operon, located at 83.5 min on the E. coli chromosome. The unc operon actually consists of nine genes which are transcribed in the order uncl, -B, -E, -F, -H, -A, -G, -D, and -C, corresponding to protein i and subunits a, c, b, 8, a(, y, P, and r, respectively. The role of protein i is currently not known, but it has been shown that this protein is not required for the activity or biosynthesis of the ATPase complex (11,28).Each gene exists in a single copy within the operon, and the operop is transcribed into a single polycistronic mRNA, yet the subunits encoded by these genes exist in different numbers in the assembled complex. Particularly intriguing is the stoichiometry of the a and c subunits of the Fo sector, which has been determined to be 1:10 (7). This unusual stoichiometry raises questions about the regulation of gene expression. Results from studies on the synthesis of ATPase polypeptides in vivo using minicells, in vitro in a transcription-translation system, or in UV-irradiated X unc lysogens indicate that the a and c subunits are synthesized differentially (4, 18). It appears that in order for the subunits to be synthesized in the appropriate relative amounts, there must be some sort of regulation, probably at the level of translation, which would allow for the expression of uncE to * Corresponding author. t Present address: E. I. du Pont de Nemours & Co., Inc., Wilmington, DE 19898. increase over that of uncB. The exact mechanism of control over the apparent differences in expression exhibited by these genes is currently unknown. McCarthy and coworkers, however, have demonstrated that an RNA sequence within the translational initiation region of uncE causes increased synthesis of the c subunit (15) and can also increase the expression of other genes when located within the translation initiation region of those genes (16). This sequence, therefore, appears to enhance expression of uncE.This research was aimed at quantitating the in vivo expression of the first three genes of the E. coli unc operon. A series of lacZ protein fusions were constructed in uncI, uncB, and uncE, and the relative level of expression of each gene was determined by comparin...

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“…5. Under these conditions, the presence of high SecA levels in the strains containing the secA gene on a multicopy plasmid resulted in less severe protein export defects when either sodium azide or the maIE-lacZ fusion (data not shown) was used to create (24,25,28). However, there is an additional UUAACU sequence beginning 13 nucleotides downstream of the secA initiation codon (38) which could have promoted a high level of SecA protein synthesis in these mutants.…”

Section: Resultsmentioning

confidence: 95%

Regulation of Escherichia coli secA mRNA translation by a secretion-responsive element

1991

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The Escherichia coli secA gene, whose translation is responsive to the proficiency of protein export within the cell, is the second gene in a three-gene operon and is flanked by gene X and mutT. By using gene fusion and oligonucleotide-directed mutagenesis techniques, we have localized this translationally regulated site to a region at the end of gene X and the beginning of secA. This region has been shown to bind SecA protein in vitro. These studies open the way for a direct investigation of the mechanism of secA regulation and its coupling to the protein secretion capability of the cell.

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Enhancement of translational efficiency by the Escherichia coli atpE translational initiation region: its fusion with two human genes

Cited by 49 publications

References 20 publications

Determinants of translational initiation efficiency in the atp operon of Escherichia coli

Determinants of translational initiation efficiency in the atp operon of Escherichia coli

Use of lacZ fusions to measure in vivo expression of the first three genes of the Escherichia coli unc operon

Regulation of Escherichia coli secA mRNA translation by a secretion-responsive element

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