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Mutational analysis of Escherichia coli PepA, a multifunctional DNA-binding aminopeptidase

par Charlier, Daniel;Huysveld, Nadine;Thia-Toong, Thia-Lin;Glansdorff, Nicolas ;Kholti, Abdelaziz;Gigot, Daniel ;Maes, Dominique
Référence Journal of Molecular Biology, 302, 2, page (411-426)
Publication Publié, 2000-09

Article révisé par les pairs

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Résumé :

Escherichia coli PepA is a hexameric aminopeptidase that is also endowed with a DNA-binding activity that functions in transcription control and plasmid dimer resolution. To gain further insight into the functioning of PepA, mutants were selected on the basis of reduced repressibility of a genomic carA-lacZ fusion and studied for the various cellular processes requiring PepA, i.e. repression of the carAB operon, autoregulation, resolution of ColE1 multimers, and peptide proteolysis. The methylation status of the carAB control region was analysed in several pepA mutants and purified proteins were assayed in vitro for car operator DNA binding. This study provides a critical test of predictions advanced on the basis of the structural analysis of PepA and demonstrates the importance for DNA binding of several secondary structural elements in the N-terminal domain and near the very C terminus. By analysis of single amino acid substitutions, we could distinguish the mode of PepA action in car regulation from its action in plasmid resolution. We demonstrate that mere binding of PepA to the car control region is not sufficient to explain its role in pyrimidine-specific regulation; protein-protein interactions appear to play an important role in transcriptional repression. The multifunctional character of PepA and of an increasing number of transcriptional regulators that combine catalytic and regulatory properties, of which several participate in the metabolism of arginine and of the pyrimidines, suggests that enzymes and DNA (RNA) binding proteins fulfilling an essential primeval function may have been recruited in evolution to fulfil an additional regulatory task. (C) 2000 Academic Press.