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Multiple protein-DNA interactions in the TATAA-less mouse thymidylate synthase promoter.

The mouse thymidylate synthase (TS) promoter lacks a TATAA box and directs transcriptional initiation at multiple sites over a 60 nucleotide region. All of the sequences that are important for transcription are located within close proximity to the first initiation site. Gel mobility shift and footprinting analyses with various sequences from this region of the TS promoter identified three major protein-DNA interactions. One of these corresponds to Sp1 interacting with a nonconsensus binding site downstream of the first transcriptional initiation site. Inactivation of the binding site by site-directed mutagenesis led to a 3-fold reduction in gene expression as well as a significant change in distribution of transcriptional start sites. The proteins responsible for the other two complexes (CII and CIII) do not appear to correspond to any of the common transcription factors that have been studied previously. The CII protein binds very close to the first initiation site. Inactivation of the binding site by site-directed mutagenesis had little effect on expression. The CIII protein binds immediately upstream of CII. Inactivation of this site led to a 12-fold reduction in expression, indicating that it is important for expression of the TS gene.