Residues near the amino terminus of Rns are essential for positive autoregulation and DNA binding.
Most members of the AraC/XylS family contain a conserved carboxy-terminal DNA binding domain and a less conserved amino-terminal domain involved in binding small-molecule effectors and dimerization. However, there is no evidence that Rns, a regulator of enterotoxigenic Escherichia coli virulence genes, responds to an effector ligand, and in this study we found that the amino-terminal domain of Rns does not form homodimers in vivo. Exposure of Rns to the chemical cross-linker glutaraldehyde revealed that the full-length protein is also a monomer in vitro. Nevertheless, deletion analysis of Rns demonstrated that the first 60 amino acids of the protein are essential for the activation and repression of Rns-regulated promoters in vivo. Amino-terminal truncation of Rns abolished DNA binding in vitro, and two randomly generated mutations, I14T and N16D, that independently abolished Rns autoregulation were isolated. Further analysis of these mutations revealed that they have disparate effects at other Rns-regulated promoters and suggest that they may be involved in an interaction with the carboxy-terminal domain of Rns. Thus, evolution may have preserved the amino terminus of Rns because it is essential for the regulator's activity even though it apparently lacks the two functions, dimerization and ligand binding, usually associated with the amino-terminal domains of AraC/XylS family members.
Basturea
Georgeta N
GN
Department of Microbiology and Immunology, University of Miami Miller School of Medicine, P.O. Box 016960 (R-138), Miami, FL 33101, USA.
Bodero
Maria D
MD
Moreno
Mario E
ME
Munson
George P
GP
D001665:Binding Sites; D003432:Cross-Linking Reagents / Q000737:chemistry; D004269:DNA, Bacterial / Q000378:metabolism*; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D005976:Glutaral / Q000737:chemistry; D009154:Mutation; D010957:Plasmids / Q000235:genetics; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D015534:Trans-Activators / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
D003432:Cross-Linking Reagents; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; C108303:Rns protein, bacteria; D015534:Trans-Activators; D005976:Glutaral