Physical organization of the upper pathway operon promoter of the Pseudomonas TOL plasmid. Sequence and positional requirements for XylR-dependent activation of transcription

• Published in: Molecular & general genetics Vol. 239; no. 1-2; p. 281
• Main Authors: Abril, M A, Ramos, J L
• Format: Journal Article
• Language: English
• Published: Germany 01.05.1993
• Subjects: Bacterial Proteins - metabolism; Base Sequence; Binding Sites; DNA, Bacterial; DNA-Binding Proteins; Gene Expression Regulation, Bacterial; Molecular Sequence Data; Operon; Plasmids; Promoter Regions, Genetic; Pseudomonas - genetics; Repetitive Sequences, Nucleic Acid; Transcription Factors; Transcription, Genetic
• ISSN: 0026-8925
• DOI: 10.1007/BF00281629

The upper pathway operon of the Pseudomonas putida TOL plasmid belongs to the -12/-24 class of promoters. These promoters exhibit three regions critical for regulated transcription, namely, the -12/-24 site for RNA polymerase/sigma 54 binding, the -55/-67 region for IHF protein binding, and the -130(UAS2)/-170(UAS1) region, where two sites for XylR binding are located. The XylR-protected G residues located at -131, -139, -160 and -169 were replaced with As, and the activity of the mutant promoters was assayed after fusion to a promoterless lacZ gene. The mutation (G(-169)-->A) resulted in a 50% decrease in expression from the promoter (Pu), whereas the other three changes had no significant effect. The XylR recognition sequence UAS2 has a perfect inverted repeat (5'-ATTTN4-AAAT-3') while UAS1 shows two mismatches (5'-CCTTN4AAAT-3'). The two Cs (located at -172 and -173), which interrupt the inverted repeat, were changed as follows: C(-172)-->T; C(-173)-->A, CC(-172, -173)-->AT. Transcription activation from the mutant promoters was measured as beta-galactosidase activity after fusion to lacZ; the better the palindromic sequence, the higher the rate of transcription from Pu, with increases in activity of up to 50%. The introduction of one or two full helix turns between the IHF and the XylR binding sites did not significantly affect transcription from Pu; however, the insertion of three helix turns resulted in a drop of 90% in the activity. The non-permissive effect of insertion of three full helix turns between the IHF and XylR binding sites was not evident in an IHF- background.