Initiation of Transcription at the Human Terminal Deoxynucleotidyl Transferase Gene Promoter: A Novel Role for the TATA Binding Protein

Abstract

Control of initiation of transcription of the human terminal deoxynucleotidyl transferase (TdT) gene was investigated by using an in vitro transcription assay. The precise contribution of discrete basal promoter elements to transcription initiation was determined by testing deletion and substitution mutations. The primary element, contained within the region spanning -34 to -14 bp relative to the transcription start site, accounted for 80% of basal promoter activity. TdT promoter activity required the sequence ACCCT at -24 to -20 bp since a dramatic decrease in transcription initiation was observed after mutation of this sequence, whereas mutation of the adjacent sequence from -32 to -25 bp did not alter promoter activity. The secondary element contained sequences surrounding the transcription start site and had 20% of promoter activity. Deletion of both elements completely abolished transcription initiation. Initiator characteristics of the secondary element were revealed by using the in vitro assay: promoter sequences at the transcription start site were sufficient to direct accurate initiation at a single site. Mutation of the sequence GGGTG spanning the transcription start site resulted in loss of transcription initiation. Both the primary and secondary elements were nonhomologous to corresponding regions from the mouse TdT gene promoter. While the human basal promoter functioned in the absence of TATA consensus sequences or GC-rich SP1 binding sites, it was dependent on active TFIID. In contrast to other TATA-less promoters, purified TATA binding protein substituted for the TFIID complex and restored promoter activity to TFIID-inactivated nuclear extracts.

Human cytomegalovirus latent gene expression in granulocyte-macrophage progenitors in culture and in seropositive individuals.

“…2A). Although the region upstream of the antisense transcript lacks a consensus TATA element, potential initiator sequences (5'-CGGGGACTCTGGGGGT-GACACCAGAGAAT-3') similar to those found in the human terminal deoxynucleotidyl transferase gene (30,31) were present. The antisense transcript was confirmed to be homogeneous and unspliced by PCR using primers IEP4J and IEP1Q (Fig.…”

TBP-like Protein (TLP/TLF/TRF2) Artificially Recruited to a Promoter Stimulates Basal Transcription in Vivo

“…The promoter fragments of MLP and TdT were inserted into a five-Gal4-binding site-carrying fire fly luciferase vector (pG5luc [Promega]) to construct pMLP and pTdT, respectively. We occasionally used the human TdT promoter (Ϫ30 to ϩ30)-derived reporter plasmid phTdT (23). The spacings between a transcription initiation site and the Gal4-binding site in pMLP, pMLP(494), pTdT, and phTdT were 52, 494, 27, and 47 bp, respectively.…”

Roles of an Ets motif and a novel CACGAC direct repeat in transcription of the murine dihydrolipoamide dehydrogenase (Dld) gene

“…It is not surprising that the same gene from different species has different transcriptional initiation mechanisms. Such differences have been found also between the human and mouse terminal deoxynucleotidyl transferase promoters [30][31][32][33].…”