Transcriptional control elements and complex initiation pattern of the TATA-less bidirectional human thymidylate synthase promoter

The nucleotide sequences thai are important for transcription of the human thymidylate synthase gene were analyzed by deletion and site-directed mutag enesis of the promoter region. Deletion analyses from the 5' and 3' ends in dicated the presence of multiple positive and negative elements. The promot er had approximately the same strength in the normal or inverted orientatio n. The region between 161 and 141 nt upstream of the translational start co don was found to be both necessary and sufficient for high-level promoter a ctivity in both directions and was designated the essential promoter region . This region, which is highly conserved in human, mouse and rat TS promote rs, contains potential binding sites for Ets, Sp1, and LSF transcription fa ctors. Site directed mutagenesis of each of these elements led to large dec reases in promoter strength. However, inactivation of potential Sp1 and E2F elements adjacent to the essential promoter region led to increases in pro moter strength. The transcriptional start site pattern was analyzed by S1 n uclease protection assays of mRNA isolated from cells transiently transfect ed with TS minigenes. Multiple start sites were detected, most of which wer e between 160 and 120 nt upstream of the AUG codon. J. Cell. Biochem. 77:50 -64, 2000. (C) 2000 Wiley-Liss, Inc.