Transcript analysis reveals an extended regulon and the importance of protein-protein co-operativity for the Escherichia coli methionine repressor
Résumé
We have used DNA arrays to investigate the effects of knocking out the methionine repressor gene, metJ, on the E. coli transcriptome. We assayed the effects in the knockout strain of supplying wild-type or mutant MetJ repressors from an expression plasmid, thus establishing a rapid assay for in vivo effects of mutations characterised previously in vitro. Repression is largely restricted to known genes involved in the biosynthesis and uptake of methionine. However, we identified a number of additional genes that are significantly up-regulated in the absence of repressor. Sequence analysis of the 5' promoter regions of these genes identified plausible matches to met-box sequences for three of these, and subsequent EMSA analysis showed that for two such loci their repressor affinity is higher or comparable to the known metB operator, suggesting that they are directly regulated. This can be rationalised for one of the loci, folE, by the metabolic role of its encoded enzyme; however the links to the other regulated loci are unclear suggesting both an extension to the known met regulon and additional complexity to the role of the repressor. The plasmid gene replacement system has been used to examine the importance of protein-protein cooperativity in operator saturation using the structurally characterised mutant repressor, Q44K. In vivo, there are detectable reductions in the levels of regulation observed, demonstrating the importance of balancing protein-protein and protein-DNA affinity.
Origine : Fichiers produits par l'(les) auteur(s)