Involvement of multiple DNA elements in iron-inducible transcription of the ap65-1 gene in the protozoan parasite Trichomonas vaginalis.

A reputed iron-responsive region, which contains multiple nuclear protein-binding DNA sequences, was shown previously to regulate iron-inducible transcription of the ap65-1 gene in the protozoan pathogen, Trichomonas vaginalis. These DNA sequences include two overlapping MYB recognition elements (MRE-1/MRE-2r) and three abutted T-tract elements. Additional nuclear protein-binding DNA sequences flanking the 5' (AGTGAAGTGA) and 3' (MRE-2f) of the iron-responsive region were identified in the present study. A stable promoter assay and primer extension revealed that transcriptional activity of the ap65-1 promoter is iron inducible as well as growth related, being lowest in the early logarithmic phase and highest in the mid-logarithmic phase. Subsequent mutational analysis of individual DNA elements of the ap65-1 promoter suggests that closely spaced T-tract elements together with an intervening GAAGGAAG sequence within the iron-responsive region are most critical for regulation of overall transcriptional activity, whereas an additional AGTGAAGTGA and MRE-2f together with an upstream T-rich region are required for optimal iron-inducible activity, and the MRE-1/MRE-2r overlap is only involved in growth-related activity. These observations suggest that expression of the ap65-1 gene is dynamically regulated under various growth conditions via interactions among multiple DNA regulatory elements.