Characterization of A transcriptional Attenuator in The rpmf-Plsx-Fab Operon of Escherichia coli K-12
Fatty acids are an essential component of the phospholipids of the inner and outer membranes of Escherichia coli. The synthesis of both fatty acids and phospholipids is regulated. Synthesis increases when growth rate increases, is inhibited when starvation occurs, and the fatty acid composition of the membrane changes with growth temperature. Several genes encoding enzymes involved in membrane synthesis are located in the rpmF-plsX-fab operon. In this operon, a gene encoding a phospholipid synthetic gene of unknown function, plsX, lies just downstream of the ribosomal protein gene rpmF and upstream of five fatty acid biosynthetic genes, fabH, fabD, fabG, acpP, and fabF. The operon is also complex; transcription is initiated from at least eight promoters. In addition, some transcripts produced by the operon are cleaved by RNases while others terminate at one of three specific points at the 5' end of plsX. This work demonstrates that a weak transcriptional terminator (an attenuator) lies at the 5' end of plsX. The attenuator was localized to a 200 bp segment. Analysis of the secondary structure of the attenuator mRNA has lead to a model which includes four stem-loop structures. In this model, the plsX start codon lies within the loop of the second stem. Two tandem stems are located directly upstream of the mapped 3' endpoints. Mutational analysis shows that all four stem-loops play a role in attenuator activity. Regulation of the attenuator and the attenuator's mechanism of controlling downstream gene expression were investigated. Ribosome binding to attenuator mRNA, the PlsX protein, ppGpp concentration, and rate of lipid synthesis all appear to have no effect on attenuator activity. Interestingly, growth temperature appears to have an effect on both attenuator activity and the activity of one or more of the promoters upstream of rpmF, P1, P2, and P3. Activity of the three promoters is 4.5-fold higher at 28*C as compared with 42*C. The attenuator also appears to increase expression of downstream genes 2-fold as temperature decreases. Though the attenuator region terminates transcription, growth temperature-regulation of attenuator activity is apparently mediated by a change in stability of the mRNA. These data demonstrate that transcriptional expression of plsX is 9-fold higher at 28*C as compared with 42*C. The striking dependence on temperature of plsX expression suggests a role for PlsX in the temperature modulation of fatty acid incorporation into the membrane phospholipids.
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