Genomic arrays are being used widely in an effort to determine the global effects of a variety of stimuli upon cellular metabolism . While virtually every paper employing this technique warns against reliance on the technique alone, this caution is often abandoned in reviewing the results. We therefore thought it of interest to compare the results of microarray data on related genes from two different laboratories and to further characterize those genes newly identified in the microarray studies by conventional molecular biological techniques. Prior to the microarray studies, the transcriptional activators of Escherichia coli, MarA, SoxS and Rob were known to stimulate the expression of 17 promoters, the marA/soxS/rob regulon. The microarray studies identified 153 genes/promoters as part of the regulon, but included only 12 of the 17 previously well-characterized members. Furthermore, only 27 of the 153 were implicated by both of the studies. Since 8 of the 27 were known members of the regulon, we analyzed the remaining 19 genes identified in both studies using conventional genetic and biochemical approaches. But first, we predicted from an algorithm for the MarA/SoxS/Rob binding site that fewer than half of the 19 would be members of the regulon. We found that only 7 were regulated by MarA, SoxS or Rob. Furthermore, statistical analysis of the results led to the conclusion that there are only 30-40 promoters in the regulon and that therefore only a fraction of the 153 genes identified by microarray analysis were biologically significant with respect to the the marA/soxS/rob regulon. The mar operon consists of a complex promoter and three structural genes, marR, marA and marB. marR encodes a repressor that autoinhibits the operon by binding at two sites: one between the -35 and -10 RNAP signals; and one just downstream of the start of transcription (corresponding to the 7th to 27th nucleotide from the 5? end of the mRNA). MarA is a transcriptional activator that stimulates 30 to 40 genes thereby rendering the cell resistant to low levels of many antibiotics and superoxides. Among the promoters activated by MarA is mar itself. Thus the mar operon is analogous to someone driving with one foot on the gas pedal and one foot on the brake. It is ideally suited for rapid induction and rapid return to homeostasis when a weak stimulus (e.g. salicylate which interacts with MarR and prevents its binding to the promoter) is briefly applied. This, because MarA is extremely unstable with a half life of ~ 1 min. We have been investigating the initiation of translation of MarR. We were unable to detect MarR by Western blot in wild-type cells which means that its concentration was below 1000 to 2000 molecules per cell. Since we know that MarA is present at about 200 molecules per cell and has a half life of about 1 min we calculate that the rate of synthesis of MarA must be about 140 molecules per minute or ~4000 per generation. That MarR is present at lower concentrations must mean either that MarR is unstable, or that it is translated inefficiently. To test the latter hypothesis we constructed paired translational and transcriptional lacZ fusion plasmids containing the mar promoter with the first 30 nucleotides of the mRNA (which includes the leader and only the GTG that serves as the first codon of MarR). We found that the transcriptional fusion made about 3000 units of beta-galactosidase whereas the translational fusion made ~2, i.e. the initiation of translation from the mar start signal is over three logs less efficient than from lacZ. Next, we constructed a series of plasmids containing the same promoter fragment with the mRNA extending to nucleotide 33, 36, 39 etc. The transcriptional fusions were unaffected but when the mar sequence extended to the 45th or the 48th nucleotide in the translational fusion, the activity increased to ~90 and ~200 units, respectively. Further lengthening the transcript to the 87th or 342th nucleotide had very little additional effect. We believe the results arise from the fact that the first 27 nt of the leader can form a nearly perfect inverted repeat preventing ribosome attachment to the GTG at nt 28-30. The stem loop structure has an estimated free energy of ~ ?5 kcal/mole. When the mRNA proceeds beyond 45 nt, two double-hairpin loop structures can be formed with similar free energies, in one of which the GTG is again immediately adjacent to the first hairpin, but in the other is more available for ribosome attachment.This has led us to thinking about repressors in general. Clearly, repressors are a class of molecules that it is important for the cell to manufacture, but to do so at relatively modest levels. In those cases where the repressor is not part of the operon this could be done by regulating transcription of the repressor. But when the repressor is part of an operon this can only be achieved by regulating translation. We are examining known repressor molecules in E coli to determine: 1) whether they have a Shine-Dalgarno sequence (MarR does not, MarA does); 2) whether they initiate translation with an AUG or with an alternate codon; 3) whether they tend to use non-abundant codons; and 4) whether their leader contains an inverted repeat upstream of the initiating codon.
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