Regulation ofin vitroexpression of theEscherichia coli frdoperon: alanine and Fnr represent positive and negative control elements

Abstract

The frdABCD operon of Escherichia coli encodes the anaerobically expressed terminal electron transport enzyme, fumarate reductase. Two mutually exclusive hairpin loop structures can occur in frdmRNA just downstream of the start of the frdA cistron. The mRNA sequence involved encodes a stretch of sequence rich in Ala and uses all four of the codons for this amino acid. In vitro expression of the frdABCD operon showed that as the level of plasmid DNA was increased from 150 fmol to 225 fmol, transcription of mRNA was suddenly elevated 6.5-fold, consistent with the concept of titrating out a repressor protein. Further studies showed that the concomitant 10.9-fold increase in translation of protein was heavily biased towards the proximal end of the operon, with little or no expression of FrdC or FrdD and a ratio of FrdA:FrdB of 2.6:1. Addition of Ala to the S-30 extract caused a 6.1-fold amplification of frd messenger transcription, a 17.6-fold increase in Frd protein translation, and a balancing of the subunit ratios to 1:1:1:1. The expression of the bla gene carried on the plasmid was not affected by DNA titration or the addition of Ala. When fnr DNA was added in equimolar ratio to frdDNA the amplification of fumarate reductase expression by Ala was abolished and the ratio of subunits produced showed a high degree of polarity with or without Ala.

Assembly of Escherichia coli fumarate reductase holoenzyme

“…A plasmid bearing the frd operon with the Tn5-truncated frdD cistron was constructed from pFRD79::Td-299 (Latour and Weiner 1987) by digesting 10 pg of plasmid DNA with 36 U of BamHI for 3 h at 37°C. The digestion products were separated on a 1.2% low-melting point agarose gel in 40 mM Tris-acetate pH 8.0, 20 mM sodium acetate, 2 mM EDTA for 18 h at 50 V constant voltage.…”

“…h (Latour and Weiner 1988). The gels were fixed in 10% acetic acid, 30% methanol, dried, and autoradiographed at -70°C on Kodak XAR-5 film.…”

“…Monospecific rabbit antibodies directed against purified FrdA and FrdB were prepared as described in Latour and Weiner (1988). Soluble and membrane fractions were prepared from pulse-chase experiments as described above.…”

“…coli DLTIII The plasmid pDJLl bears the frd operon with the Tn5 insertion in frdD (Latour and Weiner 1987). It expresses FrdABC and a truncated FrdD' , and the altered holoenzyrne can assemble in the membrane like wild-type fumarate reductase.…”

“…Separation of the frdC and frdD genes on different plasmids prevented this assembly and a functional enzyme was not made although membrane attachment occurred (Condon and Weiner 1988). Truncation of FrdD by transposon insertion in the gene results in production of a soluble trimer of FrdA, B, and C accumulating in the cytoplasm, as well as a membrane bound tetramer containing the shortened FrdD' (Latour and Weiner 1987), implying a role for FrdD in the proper assembly of fumarate reductase in the inner membrane.…”