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

✍ Scribed by Shanghong Dong; Laura Lester; Lee F. Johnson

• Publisher: John Wiley and Sons
• Year: 2000
• Tongue: English
• Weight: 278 KB
• Volume: 77
• Category: Article
• ISSN: 0730-2312
• DOI: 10.1002/(sici)1097-4644(20000401)77:1<50::aid-jcb6>3.0.co;2-c

✦ Synopsis

The nucleotide sequences that are important for transcription of the human thymidylate synthase gene were analyzed by deletion and site-directed mutagenesis of the promoter region. Deletion analyses from the 5Ј and 3Ј ends indicated the presence of multiple positive and negative elements. The promoter had approximately the same strength in the normal or inverted orientation. The region between 161 and 141 nt upstream of the translational start codon was found to be both necessary and sufficient for high-level promoter activity in both directions and was designated the essential promoter region. This region, which is highly conserved in human, mouse and rat TS promoters, contains potential binding sites for Ets, Sp1, and LSF transcription factors. Site directed mutagenesis of each of these elements led to large decreases in promoter strength. However, inactivation of potential Sp1 and E2F elements adjacent to the essential promoter region led to increases in promoter strength. The transcriptional start site pattern was analyzed by S1 nuclease protection assays of mRNA isolated from cells transiently transfected with TS minigenes. Multiple start sites were detected, most of which were between 160 and 120 nt upstream of the AUG codon.