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Identification of an intragenic ribosome binding site that affects expression of the uncB gene of the Escherichia coli proton-translocating ATPase (unc) operon.

ABSTRACT: The uncB gene codes for the a subunit of the Fo proton channel sector of the Escherichia coli F1 Fo ATPase. Control of expression of uncB appears to be exerted at some step after translational initiation. Sequence analysis by the perceptron matrices (G. D. Stormo, T. D. Schneider, L. Gold, and A. Ehrenfeucht, Nucleic Acids Res. 10:2997-3011, 1982) identified a potential ribosome binding site within the uncB reading frame preceding a five-codon reading frame which is shifted one base relative to the uncB reading frame. Elimination of this binding site by mutagenesis resulted in a four- to fivefold increase in expression of an uncB'-'lacZ fusion gene containing most of uncB. Primer extension inhibition (toeprint) analysis to measure ribosome binding demonstrated that ribosomes could form an initiation complex at this alternative start site. Two fusions of lacZ to the alternative reading frame demonstrated that this site is recognized by ribosomes in vivo. The results suggest that expression of uncB is reduced by translational frameshifting and/or a translational false start at this site within the uncB reading frame.

SUBMITTER: Matten SR

PROVIDER: S-EPMC107379 | biostudies-literature | 1998 Aug

REPOSITORIES: biostudies-literature

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Identification of an intragenic ribosome binding site that affects expression of the uncB gene of the Escherichia coli proton-translocating ATPase (unc) operon.

Matten S R SR Schneider T D TD Ringquist S S Brusilow W S WS

Journal of bacteriology 19980801 15

The uncB gene codes for the a subunit of the Fo proton channel sector of the Escherichia coli F1 Fo ATPase. Control of expression of uncB appears to be exerted at some step after translational initiation. Sequence analysis by the perceptron matrices (G. D. Stormo, T. D. Schneider, L. Gold, and A. Ehrenfeucht, Nucleic Acids Res. 10:2997-3011, 1982) identified a potential ribosome binding site within the uncB reading frame preceding a five-codon reading frame which is shifted one base relative to ...[more]

PMID: 9683492

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Project description:Insulin-secretory granules isolated from a pancreatic islet-cell tumour by centrifugation on Percoll density gradients exhibited a membrane-associated Mg(2+)-dependent ATPase activity. In granule suspensions incubated in iso-osmotic media, activity was increased 2-3-fold by carbonyl cyanide p-trifluoromethoxyphenylhydrazone, the combination of valinomycin, nigericin and K(2)SO(4) or by the addition of a detergent. Permeant anions also increased Mg(2+)-dependent ATPase activity under iso-osmotic conditions when combined with K(+) and nigericin, or NH(4) (+). It was deduced that a major component of the activity was coupled to the translocation of protons into the granule interior. The granule membrane appeared poorly permeable to H(+), K(+), NH(4) (+) and SO(4) (2-) but permeable, in increasing order, to phosphate or acetate, Cl(-), I(-) and SCN(-). Like the proton-translocating ATPase of mammalian mitochondria the granule enzyme when membrane-bound was inhibited by up to 85% by tributyltin or NN'-dicyclohexylcarbodi-imide and was solubilized in a tributyltin-insensitive form after extraction with dichloromethane. It was clearly not a mitochondrial contaminant as evidence by the distribution of marker proteins on density gradients. Unlike mitochondrial activity it was insensitive to oligomycin, efrapeptin, atractyloside, azide and oxyanions. Its properties, however, were indistinguishable from those of the proton-translocating ATPase found in the chromaffin granules of the adrenal medulla. Moreover, insulin granules and chromaffin granules exhibited similar levels of activity. This indicated that in spite of the differences in their internal composition, granules from tissues involved in polypeptide and amine hormone secretion possess catalytic components in common. Only a minor role for the ATPase in amine transport in insulin granules was apparent. Rather, its presence here may relate to the process of secretory vesicle morphogenesis or to the exocytotic mechanism.

Dataset Information

Identification of an intragenic ribosome binding site that affects expression of the uncB gene of the Escherichia coli proton-translocating ATPase (unc) operon.

Publications

Identification of an intragenic ribosome binding site that affects expression of the uncB gene of the Escherichia coli proton-translocating ATPase (unc) operon.

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