Abstract
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/σ54 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 β-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/σ54 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 Pit and the architectural organization of the Pu promoter.
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Abril, M.A., Ramos, J.L. Physical organization of the upper pathway operon promoter of the Pseudomonas TOL plasmid. Sequence and positional requirements for XylR-dependent activation of transcription. Molec. Gen. Genet. 239, 281–288 (1993). https://doi.org/10.1007/BF00281629
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DOI: https://doi.org/10.1007/BF00281629