Residues 137 and 153 at the N terminus of the XylS protein influence the effector profile of this transcriptional regulator and the sigma factor used by RNA polymerase to stimulate transcription from its cognate promoter.
The 321-residue XylS and XylS1 proteins, encoded by the pWW0 and pWW53 plasmids respectively, differ in only 5 residues at positions 4, 53, 90, 137, and 153. As a result, the effector profile of XylS is wider than that of XylS1, and XylS mediates higher levels of transcription from its cognate-regulatable promoter than does XylS1. We generated a series of XylS-pWW0 mutants and found that the single mutants Asp-137-->Glu and His-153-->Asn exhibited an activation pattern different from that of the wild-type regulator. In the double-mutant XylSD137E,H153N the effector profile for benzoates was similar to that of XylS1. This suggests that these two residues are crucial for effector recognition and regulator activation to stimulate transcription. XylS-dependent transcription from its cognate promoter is mediated by RNA polymerase with sigma(32) or sigma(38), whereas XylS1 uses RNA polymerase with sigma(32) or sigma(70). We also found that point mutations at positions 137 and 153 of XylS led RNA polymerase to mediate transcription with sigma(70) rather than with sigma(38), as demonstrated by primer extension analysis in a sigma(70)-thermosensitive background proficient and deficient in sigma(38). This suggests that a positive transcriptional regulator can choose the RNA polymerase complex that mediates transcription from a given promoter.
Ruiz
Raquel
R
Department of Biochemistry, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, Apartado de Correos 419, E-18008 Granada, Spain.
Ramos
Juan L
JL
D000596:Amino Acids / Q000737:chemistry; D001216:Asparagine / Q000737:chemistry; D001224:Aspartic Acid / Q000737:chemistry; D001426:Bacterial Proteins; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry*; D004926:Escherichia coli / Q000378:metabolism; D018698:Glutamic Acid / Q000737:chemistry; D006639:Histidine / Q000737:chemistry; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D017354:Point Mutation; D011401:Promoter Regions, Genetic; D017434:Protein Structure, Tertiary; D012313:RNA / Q000378:metabolism; D015534:Trans-Activators / Q000737:chemistry*; D014158:Transcription, Genetic; D001616:beta-Galactosidase / Q000378:metabolism
D000596:Amino Acids; D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D015534:Trans-Activators; C078720:XylS protein, Pseudomonas putida; D001224:Aspartic Acid; D018698:Glutamic Acid; D006639:Histidine; D012313:RNA; D001216:Asparagine; D004247:DNA; D012321:DNA-Directed RNA Polymerases; D001616:beta-Galactosidase