PHYSICAL ORGANIZATION OF THE UPPER PATHWAY OPERON PROMOTER OF THE PSEUDOMONAS TOL PLASMID - SEQUENCE AND POSITIONAL REQUIREMENTS FOR XYLR-DEPENDENT ACTIVATION OF TRANSCRIPTION

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/sigma54 binding, the -55/-67 region for IHF protein binding, and the -139(UAS2)/-1760(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'-ATTTN4AAAT-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. The introduction of a half helix turn between the RNA polymerase/sigma54 and IHF sites also resulted in a notable decrease in transcription from Pu; however, the introduction of one full helix turn did not affect transcription from Pu. The introduction of a full helix turn on both sides of the IHF binding site reduced expression from Pu by 75%, whereas the introduction of a half helix turn on both sides decreased expression by more than 90%. Our findings help to elucidate the sequence requirements for expression from Pu and the architectural organization of the Pu promoter.