Transcriptional activation by the human cytomegalovirus immediate-early proteins: requirements for simple promoter structures and interactions with multiple components of the transcription complex
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We have utilized a number of well-defined, simple, synthetic promoters (upstream factor binding sites and TATA elements) to analyze the activation mechanisms of the human cytomegalovirus immediate-early (IE) proteins. We found that the 86-kDa IE protein (known as IEP86, IE2(559aa), or ppUL122a) can recognize and activate a variety of simple promoters, in agreement with the observation that it is a promiscuous activator. However, in the comparison of otherwise identical promoters IEP86 does have preferences for specific TATA elements (hsp70 > adenovirus E2 > simian virus 40 early) and specific upstream transcription factor binding sites (CAAT > SP1 approximately Tef-1 > ATF; no activation with AP1 or OCT). In contrast, the 72-kDa IE protein (known as IEP72, IE1(491aa), or ppUL123) alone did not significantly activate the simple promoters under our experimental conditions. However, each promoter activated by IEP86 was synergistically affected by the addition of IEP72. In addition, the 55-kDa IE protein (IEP55, a splice variant form of IE2, IE2(425aa), or ppUL122b) repeatedly had a negative effect, downregulating the activation of promoters caused by IEP86 and the synergy of IEP86 and IEP72. We show that the ability of IEP86 to activate many simple promoters correlates not only with its previously described ability to interact with the TATA-binding protein (TBP) (B. A. Furnari, E. Poma, T. F. Kowalik, S.-M. Huong, and E.-S. Huang, J. Virol. 67:4981-4991, 1993; C. Hagemeier, S. Walker, R. Caswell, T. Kouzarides, and J. Sinclair, J. Virol. 66:4452-4456, 1992; R. Jupp, S. Hoffman, R. M. Stenberg, J. A. Nelson, and P. Ghazal, J. Virol. 67:7539-7546, 1993) but also with its ability to interact with the transcription factors which bind to the upstream element of promoters it activated (e.g., SP1 and Tef-1 but not Oct-1). This ability to have multiple interactions with the promoter complex may be crucial for transcriptional activation, since the IE proteins cannot activate promoters having only a TATA element or only an upstream transcription factor binding site. In addition, we show that proteins which bind IEP86 also bind to IEP55. Thus, the negative effect on transcription noted with IEP55 may be the result of competition with IEP86 for interaction with the promoter complex. The synergy caused by IEP72 appears to be mediated by a more indirect mechanism. This is suggested by our observation that IEP72 could not bind to any of the proteins tested (TBP, Tef-1, or Oct-1) or to IEP86.Keywords:
TATA box
CAAT box
Transcription
We studied the response of simple synthetic promoter regions to transactivation by the adenovirus early region 1A (E1A) protein. Binding sites for one or two host cell transcription factors were substituted for the E1B promoter region in reconstructed virus mutants, and the response to E1A transactivation was assayed during the early phase of infection. We found that a single CREB/ATF binding site resulted in a surprisingly strong promoter which responded to E1A. A CREB/ATF binding site placed upstream of the E1B TATA box behaved much like the wild-type E1B promoter, which is composed of a single Sp1 binding site plus a TATA box. A single E2F binding site resulted in an extremely weak promoter which did not respond to E1A, much like a single Sp1 site. Two E2F sites in an inverted orientation with the same spacing as in the adenovirus type 2 E2 early promoter produced a strong, E1A-responsive promoter. Substitution of the E4 TATA box region for the E1B TATA box region produced a promoter about five times stronger than the wild-type E1B promoter in the absence of E1A. However, the E4 TATA box substitution did not respond significantly to E1A transactivation. These results directly demonstrate that many different transcription factor binding sites, including the E1B TATA box, a CREB/ATF binding site, and two E2F sites, can mediate E1A transactivation. Other transcription factor binding sites cannot mediate an E1A response; these other sites include the E4 TATA box, a single Sp1 binding site, and a single E2F binding site. Implications of these findings for the mechanism of E1A transactivation are discussed.
TATA box
E2F
TATA-binding protein
CAAT box
DNA binding site
Activating transcription factor
TATA-Box Binding Protein
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Nuclear factor I (NFI) is composed of a family of site-specific DNA-binding proteins which recognize a DNA-binding site with the consensus sequence TGGC/A(N)5GCCAA. Binding sites for NFI have previously been shown to stimulate mRNA synthesis in vitro when present upstream of the TATA box of the adenovirus major late promoter (AdMLP). We have examined the effect of NFI-binding sites on transcription in vivo in transiently transfected HeLa and COS cells. An NFI-binding site isolated from the human genome activated expression from the minimal AdMLP in vivo in both the absence and presence of the simian virus 40 enhancer. A point mutation that decreased NFI binding affinity for the site in vitro reduced expression to near the basal level of the AdMLP. Several NFI-binding sites which differed in their spacer and flanking sequences were tested for their ability to activate expression in vivo. The ability of these sites to activate expression correlated with the strength of NFI binding in vitro. An NFI-binding site stimulated expression equally well when placed from 33 to 65 bp upstream of the TATA box. However, expression dropped to basal levels when the site was located from 71 to 77 bp upstream of the TATA box. These studies indicate that an NFI-binding site in this chimeric promoter activates expression in vivo only if located within a critical distance of the TATA box.
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Upstream activating sequence
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Chalcone synthase(CHS),coded by the chs gene super-family,is a key enzyme in flavonoid biosynthesis.Two independent promoters was isolated for chsA,named PchsA-L(550 bp) and PchsA-S(354 bp)(GenBank accession number EF199747 and EF199748 respectively),from the genomic DNA of Petunia hybrida.PchsA-L differs with PchsA-S mainly in that PchsA-L has a 182 bp fragment from 88～269 bp,and the sequence 103～201 bp has the characteristics of a typical intron.Both promoter sequences contain conserved sequences of TATA box,CCAAT box,cap site(CCATAA),and the flower-specific promoter sequences TACPyAT box,anther box(TAGAAGTGACAGAAAT),G-box(CACGTG),box1 element(ATGTCACGTGCCATC) and box2 element(TGTGTTGAAGGTTTGCTA).The petunia plant used for promoter cloning was a diploid with 14 chromosomes.Southern blotting showed that both promoters had multiple copies in the genome.The two promoters segregated in the offspring but the segregation did not meet the ratio of 1∶2∶1.qRT-PCR analysis showed no significant difference in chsA gene expression in non-UV-treated and UV-treated floral organ of plants with one or both promoters.The expression of chsA in the UV-treated seedling leaves was increased compared with UV-treated floral organ,and PchsA-L-driven chsA expression in the UV-treated seedling leaves was very significantly increased than that driven by PchsA-S while there was no chsA gene expression in non-UV-treated seedling leaves.The results show the presence of two independent promoters PchsA-L and PchsA-S for chsA in the petunia genome;the 182 bp intron-like sequence in PchsA-L promoter could significantly increased chsA gene expression in UV-treated seedling leaves.
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Journal Article Upstream regulatory regions required to stabilize binding to the TATA sequence in an adesovirus early promoter Get access Joseph Garcia, Joseph Garcia Division of Hematology-Oncology11-242 Louis Factor BuildingDepartment of Medicine, UCLA School of Medicine and Jonnson Comprehensive Cancer CenterLos Angeles, CA 90024, USA Search for other works by this author on: Oxford Academic PubMed Google Scholar Food Wu, Food Wu Division of Hematology-Oncology11-242 Louis Factor BuildingDepartment of Medicine, UCLA School of Medicine and Jonnson Comprehensive Cancer CenterLos Angeles, CA 90024, USA Search for other works by this author on: Oxford Academic PubMed Google Scholar Richard Gaynor Richard Gaynor Division of Hematology-Oncology11-242 Louis Factor BuildingDepartment of Medicine, UCLA School of Medicine and Jonnson Comprehensive Cancer CenterLos Angeles, CA 90024, USA Search for other works by this author on: Oxford Academic PubMed Google Scholar Nucleic Acids Research, Volume 15, Issue 20, 26 October 1987, Pages 8367–8385, https://doi.org/10.1093/nar/15.20.8367 Published: 26 October 1987 Article history Received: 24 June 1987 Revision received: 01 September 1987 Accepted: 01 September 1987 Published: 26 October 1987
TATA box
DNA binding site
CAAT box
DNA footprinting
Upstream activating sequence
Footprinting
A-site
TATA-binding protein
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[Objective] The ras promoters were cloned from genomic DNA of Auricuralia auricular so as to provide promoters for breeding better species by genetic technology.[Method] PCR was used to clone promoters with the genomic DNA of A.auricular strain YBS-3 as template,and then the sequences of four ras promoters were obtained.The analysis of promoter sequences was made by three promoter analysis software:Promoter prediction,Place and TFSEARCH ver.1.3.[Result] Four fragments contained core elements of promoter including TATA-box and CAAT-box,and many other important cis-elements such as GATA BOX,GCGC BOX,CCAAT BOX1,etc.At the same time,there was at least one transcriptional start site in these sequences.And several transcription factor binding sites were detected in these sequences.[Conclusion] Four promoter fragments were all having promoter function theoretically.
TATA box
CAAT box
Cloning (programming)
Transcription
DNA binding site
genomic DNA
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Nuclear factor I (NFI) is composed of a family of site-specific DNA-binding proteins which recognize a DNA-binding site with the consensus sequence TGGC/A(N)5GCCAA. Binding sites for NFI have previously been shown to stimulate mRNA synthesis in vitro when present upstream of the TATA box of the adenovirus major late promoter (AdMLP). We have examined the effect of NFI-binding sites on transcription in vivo in transiently transfected HeLa and COS cells. An NFI-binding site isolated from the human genome activated expression from the minimal AdMLP in vivo in both the absence and presence of the simian virus 40 enhancer. A point mutation that decreased NFI binding affinity for the site in vitro reduced expression to near the basal level of the AdMLP. Several NFI-binding sites which differed in their spacer and flanking sequences were tested for their ability to activate expression in vivo. The ability of these sites to activate expression correlated with the strength of NFI binding in vitro. An NFI-binding site stimulated expression equally well when placed from 33 to 65 bp upstream of the TATA box. However, expression dropped to basal levels when the site was located from 71 to 77 bp upstream of the TATA box. These studies indicate that an NFI-binding site in this chimeric promoter activates expression in vivo only if located within a critical distance of the TATA box.
TATA box
CAAT box
DNA binding site
Upstream activating sequence
Ccaat-enhancer-binding proteins
Transcription
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2541 eukaryotic promoter sequences that had been validated by test were downloaded and the number of TATA-box,GC-box and CAAT-box and its distribution conditions in the promoters were analyzed by bioinformatics method.The results showed that 23.85% eukaryotic promoter sequences had at least one TATA-box and TATA-box distributed mainly in the area 23～128 bp ahead of transcription initiation site.47.30% eukaryotic promoter sequences had at least one GC-box and GC-boxes were concentrated in the area 23～128 bp ahead of transcription initiation site.42.35%eukaryotic promoter sequences had at least one CAAT-box and CAAT-boxes were concentrated in the area 51～159 bp ahead of transcription initiation site.The results indicated that the position of TATA-box in eukaryotic promoters was relatively fixed and it might play an important role in the correct initiation of gene transcription.But GC-box and CAAT-box were widely distributed and their number was obviously more than that of TATA-box.
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The availability of sequence specificities for a substantial fraction of yeast's transcription factors and comparative genomic algorithms for binding site prediction has made it possible to comprehensively annotate transcription factor binding sites genome-wide. Here we use such a genome-wide annotation for comprehensively studying promoter architecture in yeast, focusing on the distribution of transcription factor binding sites relative to transcription start sites, and the architecture of TATA and TATA-less promoters. For most transcription factors, binding sites are positioned further upstream and vary over a wider range in TATA promoters than in TATA-less promoters. In contrast, a group of 'proximal promoter motifs' (GAT1/GLN3/DAL80, FKH1/2, PBF1/2, RPN4, NDT80, and ROX1) occur preferentially in TATA-less promoters and show a strong preference for binding close to the transcription start site in these promoters. We provide evidence that suggests that pre-initiation complexes are recruited at TATA sites in TATA promoters and at the sites of the other proximal promoter motifs in TATA-less promoters. TATA-less promoters can generally be classified by the proximal promoter motif they contain, with different classes of TATA-less promoters showing different patterns of transcription factor binding site positioning and nucleosome coverage. These observations suggest that different modes of regulation of transcription initiation may be operating in the different promoter classes. In addition we show that, across all promoter classes, there is a close match between nucleosome free regions and regions of highest transcription factor binding site density. This close agreement between transcription factor binding site density and nucleosome depletion suggests a direct and general competition between transcription factors and nucleosomes for binding to promoters.
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CAAT box
TATA-binding protein
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General transcription factor
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TATA box
RNA polymerase II
CAAT box
Transcription
TATA-Box Binding Protein
TAF2
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CAAT box
TATA box
Upstream activating sequence
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Citations (68)