Control of transcription initiation in vitro requires binding of a transcription factor to the distal promoter of the ovalbumin gene.

Abstract

We used a cell-free HeLa cell transcription system to identify and characterize transcription factors and the promoter elements that they recognize in RNA polymerase II-transcribed genes. Deletion of the region (-71 to -83) containing the GTCAAA direct repeat resulted in a marked decrease of specific transcription of the ovalbumin gene; transcription could be competed with DNA fragments containing this sequence. Furthermore, DNase I footprinting identified a protein-binding site including this direct repeat with crude extracts and one of the partially purified protein fractions required for transcription. We propose that a soluble factor activates transcription through binding to the direct repeat of GTCAAA sequence upstream from the ovalbumin gene.Recombinant DNA methodology and the techniques of gene transfer have facilitated the characterization of promoters for RNA polymerase II-transcribed genes. The efficiency of mutated promoters in directing accurate initiation has been tested in vivo and in vitro (1,2,10,32). These experiments suggested the existence of distinct promoter elements upstream from the initiation site of transcription. The TATA box, a highly conserved homology among mRNA coding genes and located at -24 to -32 base pairs from the initiation site, is essential for efficient and accurate initiation of most genes both in vivo and in vitro (2, 32). In addition, sequences upstream from the TATA box have been shown to be required in vivo (32) and in vitro (20,22,24,27,29,40,41) for efficient transcription of many eucaryotic genes. This is clearly the case for the ovalbumin gene in vivo (28). We previously showed that accurate initiation of the ovalbumin gene transcription in vitro requires the TATA homology (39, 42). We demonstrate here that sequences upstream from the TATA box are important for quantitative transcription of the ovalbumin gene in crude extracts from HeLa cells. This upstream promoter element is located approximately 80 base pairs from the cap site and includes a direct repeat of GTCAAA sequence. Since this region was originally defined by Benoist et al. (1) and Efstratiadis et al. (11) as the CAAT-box region, we tentatively designate the direct repeat sequence as the CAAT box for reference purposes.The requirement for a distal promoter element was initially indicated by 5' deletion mapping. To test whether a transcription factor interacts with this promoter element, we performed transcription competition assays and found that crucial sequences for the binding of a transcription factor include the CAAT box. We have shown previously by exonuclease footprinting that a protein which binds to this region of the ovalbumin gene is present in HeLa cell fractions (13). We now confirm and extend this footprinting analysis using DNase I with either crude nuclear extracts or the partially purified fractions (38) required for transcription in reconstituted systems. These results suggest that the upstream element delineated by the functional transcription assays is the specific binding site fo...

Nuclear Receptor Involvement in the Regulation of Rat Cytochrome P450 3A23 Expression

“…5A) nucleotides, respectively, that specifically bind with COUP-TF but not HNF-4 (38). The COUP oligonucleotide corresponds to the natural COUP-TFI binding site found in the chicken ovalbumin promoter and comprises a DR2 (39). These oligonucleotides competed with labeled DexRE-2 for complex formation as would be predicted if COUP-TF were the binding protein (Fig.…”

Multiple 5'-flanking regions of the human alpha-skeletal actin gene synergistically modulate muscle-specific expression.

“…This discrepancy appears to result from the impact of reduced transcriptional activity directed by the benchmark P-actin promoter in L8 cells relative to the activity of the baseline control plasmid, pHCAOCAT (Table 1 [32,51,58,61], human, rabbit, and mouse P-globin [25,35], a-globin [57], fibroin [56], immunoglobulin kappa light chain [44]), which are normally expressed only in differentiated tissues. The 5' ends of the in vitro products have been identical to the in vivo 5' ends in all cases.…”

COUP-TF Upregulates NGFI-A Gene Expression through an Sp1 Binding Site

“…COUP-TF was first described as an essential factor for the transcription of the chicken ovalbumin gene (42) and was subsequently cloned from many other organisms (for a review, see reference 58). The amino acid sequence conservation of COUP-TFs in different species suggests that COUP-TFs play important roles in vivo.…”