Evidence against translational repression by the carboxyltransferase component of Escherichia coli acetyl coenzyme A carboxylase

Abstract

In Escherichia coli, synthesis of the malonyl coenzyme A (malonyl-CoA) required for membrane lipid synthesis is catalyzed by acetyl-CoA carboxylase, a large complex composed of four subunits. The subunit proteins are needed in a defined stoichiometry, and it remains unclear how such production is achieved since the proteins are encoded at three different loci. Meades and coworkers (G. Meades, Jr., B. K. Benson, A. Grove, and G. L. Waldrop, Nucleic Acids Res. 38:1217-1227, 2010, doi:http://dx.doi.org/10.1093/nar/gkp1079) reported that coordinated production of the AccA and AccD subunits is due to a translational repression mechanism exerted by the proteins themselves. The AccA and AccD subunits form the carboxyltransferase (CT) heterotetramer that catalyzes the second partial reaction of acetyl-CoA carboxylase. Meades et al. reported that CT tetramers bind the central portions of the accA and accD mRNAs and block their translation in vitro. However, long mRNA molecules (500 to 600 bases) were required for CT binding, but such long mRNA molecules devoid of ribosomes seemed unlikely to exist in vivo. This, plus problematical aspects of the data reported by Meades and coworkers, led us to perform in vivo experiments to test CT tetramer-mediated translational repression of the accA and accD mRNAs. We report that increased levels of CT tetramer have no detectable effect on translation of the CT subunit mRNAs.

FIG 1

The acetyl-CoA carboxylase (ACC) reaction. The synthesis of malonyl-CoA is carried out in two distinct partial reactions. (A) The reaction is initiated by the carboxylation of the covalently bound biotin of the accB-encoded biotin carboxyl carrier protein (BCCP) by the accC-encoded biotin carboxylase (BC). (B) In the second partial reaction, the carbonyl group is transferred from the biotin moiety of the carrier protein to acetyl-CoA by the carboxyltransferase (CT) component (encoded by accA and accD) to give malonyl-CoA.

FIG 2

Experimental design. (A) The E. coli MG1655 host strain carried three compatible plasmids. The color of the promoter symbol denotes the RNA polymerase that transcribes the gene(s) of interest. These plasmids were a low-copy-number kanamycin-resistant plasmid encoding the two CT proteins under the control of the phage T5 promoter (in blue; pACS281), a medium-copy-number chloramphenicol-resistant plasmid encoding phage T7 RNA polymerase under the control of the arabinose (paraBAD) promoter (in blue; pTARA), and a low-copy-number plasmid encoding either AccA or AccD under the control of the phage T7 promoter (in red; pACS243 or pACS245, respectively). The phage T5 and araBAD promoters are recognized only by the rifampin-sensitive RNA polymerase of the E. coli host, whereas the phage T7 promoter is recognized only by the rifampin-insensitive T7 RNA polymerase. Cultures carrying the three plasmids were grown to mid-exponential phase and split in half. One half received IPTG to induce CT overexpression, whereas the other half received no inducer (low expression level denoted by the small dot size). Following growth to allow CT accumulation, IPTG was removed and the cells were resuspended in fresh medium and allowed to resume growth. Rifampin was then added to block utilization of E. coli promoters, and the cultures were incubated to allow degradation of nascent host mRNAs. (B) Following incubation, a small portion of each culture was briefly labeled with [³⁵S]methionine to monitor translation (the proteins are denoted by the chain of multicolored shapes) of the accA or accD mRNA produced by T7 RNA polymerase to test whether or not the presence of higher concentrations of CT tetramers (the small tetrameric shapes) bound the mRNA and inhibited its translation. The labeled proteins were then separated by SDS-PAGE, and the band intensities were quantitated using a phosphoimager and cognate software. The cartoon gels at the bottom of the figure give the possible experimental outcomes (AccA and AccD are of similar size). The portions of the cultures that were not exposed to [³⁵S]methionine were used to assay CT overexpression by Western blotting.

FIG 3

Overproduction of the wild-type CT tetramer and its effect on translation of the accA or accD mRNA. (A to C) The upper phosphorimager scans of the gels show the results of [³⁵S]methionine labeling (³⁵S Met), whereas the lower gels are results from Western blotting assays of CT overexpression. The lanes in each panel of the two gels are in strict correspondence. The host strain was E. coli K-12 MG1655. Note that the loss of resolution resulting from the Western blotting transfer step obscures separation of the similarly sized AccA (35.2 kDa) and AccD (33.2 kDa) subunits. Both proteins are present as demonstrated by the high CT activity of the purified protein. (A) Upper gel, [³⁵S]methionine labeling of AccA (lanes 5 to 7) or AccD (lanes 8 to 10) expressed from the mRNAs transcribed from the T7 promoter in the presence or absence of host RNA polymerase-expressed CT overproduction. The labeling of either AccA or AccD with [³⁵S]methionine in the presence of rifampin was following cultures left without induction or induced for CT overproduction with IPTG (100 or 500 μM). The plasmid used for CT overproduction was pACS280, whereas the T7 promoter plasmids encoding AccA and AccD were pACS243 and pACS245, respectively. Lower gel, Western blot assays of CT overexpression. Extracts of the unlabeled cultures performed in parallel with the [³⁵S]methionine-labeling experiments (see Materials and Methods). CT was expressed from plasmid pACS280 without or with induction by IPTG, as given. Either the accA or the accD T7 expression plasmid (pACS243 or pACS245, respectively) was present for assay of the encoded CT subunit expression by [³⁵S]methionine labeling. The band intensity responses of the Western blot to differing levels of purified AccA and the relative CT overexpression in the absence or with IPTG induction are shown. Lanes 1 to 3 show quantitation standards of decreasing quantities of purified AccA (3.31, 0.882, and 0.441 μg). Lane 4 shows a control AccA protein produced from a T7 transcript that encodes a protein containing a thrombin tag adjacent to the pentahistidine tag that for unknown reasons results in a protein that migrates more slowly than the AccA species encoded by the CT expression plasmid. Basal expression from the T7 promoter produced the AccA subunit present in lanes 5 to 7 (migrating slower than the CT complex) and the AccD subunit present in lane 8 (which migrates similarly to the CT complex and is thus obscured in lanes 9 and 10). These subunits are unable to form CT tetramers due to the lack of overexpression of the other subunit. Comparison of the intensities of the tetramer bands to that of the “missing” subunit (that not present in the T7 expression plasmid) in the lanes that lacked IPTG gives a rough estimate of the extent of overproduction. In the uninduced samples, tetramer expression by the host RNA polymerase was below the level of detection, and thus direct comparison with the subunit expressed from the T7 expression plasmid gave minimum ratios of overexpression for lanes 6, 7, 9, and 10 of >7-fold, 14-fold, 14-fold, and 27-fold, respectively. (B) The effects of overproduction of this mutant CT on translation of the accA (lanes 5 and 6) or accD (lanes 7 and 8) mRNAs. This experiment is essentially identical to that of panel A except that the CT expressed was from plasmid pACS281, which encoded an AccD subunit that lacked zinc finger function due to two cysteine-to-alanine substitutions of residues 27 and 30 (12). The IPTG concentration was 100 μM, which gave minimum overexpression levels of 7-fold (accA experiment) and 9-fold (accD experiment). Lanes 1 to 3 of the Western blot shows the purified AccA standard (3.31, 0.882, and 0.441 μg) as well as AccA expressed by T7 polymerase in lane 4. (C) This experiment assayed translation of the accA (lanes 5 and 6) or accD (lanes 7 and 8) mRNAs in the absence of CT overexpression (the empty vector plasmid, pACS285, was used), and therefore the proteins labeled with [³⁵S]methionine are the same as those detected by Western blotting. The four AccA quantitation standard lanes were loaded with 3.31, 0.882, 0.441, or 0.221 μg of protein (lanes 1 to 4). (D) The relative changes in intensity of the ³⁵S-labeled protein bands from uninduced samples and those induced with 100 μM IPTG are given. No significant decreases in ³⁵S-labeled protein levels were found. The intensity values were 1.16 ± 0.14, 1.1 ± 0.03, 1.1 ± 0.09, 1.02 ± 0.066, 1.09 ± 0.072, and 0.96 ± 0.11. These data were obtained from three biological replicates and are given as standard errors. D*A denotes the CT tetramer containing the mutant (C27A C30A) AccD protein. In the figure, WB denotes Western blot.