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

Abstract

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...

Detection and localization of thei protein inEscherichia coli cells using antibodies

“…These results are in accord with those obtained with minicells harboring pl.zsmids which encode the uncI gene with its natural translational initiation region, thereby confirming the conclusions drawn with respect to the molecular weight and the N-terminal sequence of the chromosome-encoded protein [10,1 I]. The relatively weak immunolabeling of the i protein in preparations of Fo and F~Fo compared to a labeling of the Fo subunits (data not shown) lends further support to the notion that this protein is synthesized in substroichiometric amounts [11][12][13].…”

“…The synthesis level of the i protein was found to be comparable to that of subunit a or 7/ [9], which is, however, in sharp contrast to the in vivo situation, since the i protein has so far not been detected in intact cells or in preparations of the ATP synthase complex. Furthermore, from investigations on translational initiation carried out with the individually subcloned tmcl gone [10,I1] or with uncL':lacZ fusions [12,13] it was concluded that the protein is either synthesized on a substoichiometric level in comparison to all other ATP synthase subunits or it is not expressed at all [12]. …”

Effects of the uncI gene on expression of uncB, the gene coding for the a subunit of the F1F0 ATPase of Escherichia coli

“…Plasmids pKS102, pDKWH103, pKS103, pKS104, carrying the uncB'-'lacZ fusion genes, and pKS105, carrying the uncE'-'lacZ fusion gene, have all been described previously [21]. The fusion gene in pDKWH 107 was constructed by first cloning the 617 base pair BamHI fragment within uncB into M13mpl8 to create pDKWH1301.…”

Transcriptional regulation of the proton-translocating ATPase (atpIBEFHAGDC) operon of Escherichia coli: control by cell growth rate

“…All of the genes appear to be cotranscribed from a single promoter to give a 7-kb mRNA that terminates just following the atpC gene (9,13,28). Numerous investigations have documented the differential translation of the atp genes such that each subunit of the ATPase is produced in the appropriate amount for assembly into the mature F 0 F 1 complex (1,18,19,27,29,31,35). This process involves an ordered endonucleolytic processing of the atp message to yield several stable intermediates that are then differentially translated.…”