The mouse thymidylate synthase promoter lacks a TATA box and initiates transcription at many sites across a 90-nucleotide initiation window. We showed previously that wild-type promoter activity is maintained with a promoter that extends only 13 nucleotides upstream of the first start site. G/A-rich and G/C-rich promoter elements were identified in the vicinity of the first transcriptional start site. The goals of the present study were to determine whether there are additional promoter elements in the initiation window and to determine why transcription initiates across such a broad region. Minigenes containing a variety of substitution, deletion, and insertion mutations in the promoter region were transfected into cultured cells, and the effects on expression and the pattern of start sites were determined. The results indicate that there are no additional promoter elements downstream of the G/C box. The boundaries of the transcription window are established by elements near the 5' end of the window, whereas the pattern of start sites is determined by sequences within the window. The promoter lacks an initiator element. When an initiator element was inserted, transcription initiated predominantly at the position directed by the initiator when it was inserted within the initiation window but not when it was inserted immediately upstream of the window.The promoters of protein-coding genes in higher eukaryotes generally consist of a TATA motif in combination with one or more binding sites for transcriptional regulatory proteins. Transcriptional initiation by RNA polymerase II occurs at a single site 25 to 30 nucleotides downstream of the TATAA sequence. The TATA motif is the binding site for the general transcription factor TFIID (29,34). This factor promotes the assembly of the transcription initiation complex, which includes many additional factors as well as RNA polymerase II (3, 39). The TATA box also plays an important role in the selection of the site of transcriptional initiation, since alterations in the distance between the TATA box and the normal start site can lead to the creation of a new initiation site approximately 30 nucleotides downstream of the TATA box (24).Although most class II promoters contain a TATA box, there are many examples of promoters which initiate transcription at a unique site but which lack an A/T-rich sequence 30 nucleotides upstream of the initiation site (13,16,41). A special DNA sequence at the transcriptional start site, which has been designated the initiator element, is responsible for specifying the transcriptional start site in such promoters (2,4,26,41,47). Initiator elements have also been identified in some TATA-containing promoters (6, 30). Many initiator elements serve as binding sites for trans-acting factors and are able to function autonomously to initiate transcription (4,26,32,33,36,40,42).Finally, there are several examples of TATA-less promoters that have multiple, widely dispersed sites of transcriptional initiation (14,15,27,35). Little is known about the me...