The human thymidine kinase gene promoter. Deletion analysis and specific protein binding

Abstract

We report a functional analysis of the human thymidine kinase (tk) gene promoter. We have linked the tk promoter to the chloramphenicol acetyltransferase (CAT) gene to allow direct measurement of promoter strength by assaying chloramphenicol acetyltransferase enzyme activity after transfection into mouse L cells. Putative transcription elements have been identified by deletion and mutation analysis of this promoter. The promoter relies primarily on two "CCAAT" elements and a series of "GC" elements found farther upstream. Two-thirds of promoter activity is maintained by a construct containing 139 base pairs of sequence upstream of the initiation of transcription that contains only one GC and one of the CCAAT elements. In addition, an evolutionary comparison identifies two highly conserved promoter elements: the -40 CCAAT element and a "TATA" element located at -21. We have further characterized both CCAAT elements using a mutational as well as protein binding analysis. From this study we have determined that both the -70 and -40 CCAAT elements bind strongly to the same factor, with a slightly higher affinity for the -40 CCAAT. Competition studies suggest that the CCAAT factor that binds to this promoter is homologous to protein nuclear factor Y, which binds to the major histocompatibility complex class II E alpha gene promoter. In addition, either CCAAT element is capable of supplying almost as much promoter strength as is supplied in the presence of both.