Haq, M.A.; (2007) The AmiR/RNA interaction of the amidase operon regulatory system of Pseudomonas aeruginosa. Doctoral thesis , University of London.

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Abstract

Expression of the amidase operon of Pseudomonas aeruginosa is regulated by the AmiC and AmiR proteins using a version of the bacterial two-component signal transduction system. The negative regulator, AmiC is the amide ligand sensor and regulates activity of AmiR by a steric hindrance mechanism. AmiR, the response regulator functions as a transcription antitermination factor. Once released from the AmiC/AmiR complex, AmiR binds to the operon leader transcript to prevent formation of the rho-independent terminator thus allowing transcription of the entire operon. AmiR has a CheY-like N-terminal response regulator receiver domain without the conserved phosphate-acceptor residues and a long coiled-coil C-terminal domain terminating in a three-helix bundle ANTAR domain. Within this family are a number of highly conserved residues. These residues have been changed by site-directed mutagenesis and the mutants tested for antitermination activity by amidase assay. Random mutagenesis has also revealed key residues in this domain, changes which led to reduced antitermination efficacy. To define the minimal transcript length sufficient for AmiR antitermination, constructs have been made and analysed containing leader region mutations and deletions. The results show that the encoded leader ORF has no biological function and that sequences upstream of the previously defined L recognition region are not necessary for AmiR-dependent antitermination. The ANTAR domain has been expressed and purified as a cleavable, GST tagged fusion protein in E. coli. Dynamic light scattering data and gel filtration elution profiles of ANTAR suggests this domain is a dimer in solution. The ANTAR-RNA interaction was investigated in vitro using band shift assays. Radiolabeled in vitro transcribed ami leader RNA was incubated with the ANTAR protein under various buffer conditions. The mixtures were analysed by PAGE to monitor the formation of protein-RNA complexes. Amidase assays were also performed with the ANTAR domain for analysis of antitermination activity in vivo. These results show that the ANTAR domain alone is not sufficient for antitermination in vivo or binding to the RNA in vitro.

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