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Human DNA TATA boxes and transcription initiation sites. A statistical study.

A statistical study of "TATA", or Hogness, boxes and transcription initiation sites in human DNA protein-coding genes is presented. The range over which the two signal sequences extend is assessed, and their information content is evaluated. The results are compared with those obtained from "general eukaryotic" samples (including sequences from various eukaryotic species and viruses). Statistically significant differences are found for both signals, and are especially noticeable for transcription initiation sites. The possibility that the latter may contain sufficient information to resolve the remaining ambiguity in the position of the transcription start point once a TATA box has been located is explored.

Base Sequence↗

Functional modules important for activated expression of early genes of herpes simplex virus type 1 are clustered upstream of the TATA box.

Functional analysis of two promoters controlling early herpes simplex virus type 1 (HSV-1) transcripts encoding the UL37 and UL50 (dUTPase) proteins are described in this report. Transcripts expressed under the control of these promoters were found to be expressed early regardless of the position of the transcription unit within the viral genome. Despite this, wt dUTPase mRNA was 6-10 times more abundant than the UL37 transcript both in wt and recombinant viruses. This same difference in transcript abundance was seen when a reporter gene (beta-galactosidase) was controlled by the two promoters in recombinant viruses in the heterologous glycoprotein C (gC) locus. Thus, both the kinetics and relative abundance of UL50 and UL37 transcripts are a direct function of their respective promoter regulatory elements. Characterization of mutated UL37 and UL50 promoters in recombinant viruses showed that the functional modules important for expression from these promoters are concentrated upstream of the transcription start site; however the extent and composition of these modules in terms of the cis-acting elements they contain was different for each. For the UL37 promoter, both a HiNF-P factor binding site (-53 to -58 bp) and the TATA homology (-22 to -27) were required for any detectable expression, while an Sp1 binding site at -123 augmented this but was not absolutely required. In contrast, the only functional elements crucial for expression from the UL50 promoter were the TATA box (-25 to -31) and an Sp1 binding site at -117 bp relative to the cap site. Despite differences in detail, when the functional architecture of these two early promoters were compared to the extensively characterized HSV-1 thymidine kinase (UL23) promoter, class-specific similarities are clearly apparent.

Animals↗

Mutational analysis of sequences downstream of the TATA box of the herpes simplex virus type 1 major capsid protein (VP5/UL19) promoter.

Transient expression assays with the herpes simplex virus type 1 (HSV-1) promoter/leader controlling the beta gamma (leaky-late) VP5 (UL19) mRNA encoding the major capsid protein showed that no more than 36 to 72 bases of VP5 leader are required for full-level expression. Constructs lacking the viral leader and the transcription initiation site expressed the reporter gene at about 20% of the maximum level. We confirmed this observation by using recombinant viruses in which VP5 promoter/leader deletions controlling the bacterial beta-galactosidase gene were inserted into the nonessential glycoprotein C (UL44) locus of the genome. Sequences within +36 are required for full-level expression, and removal of all leader sequences including the cap site resulted in a 10-fold decrease in reporter mRNA accumulation. The removal of the leader sequence had a measurable effect upon the kinetics of reporter mRNA accumulation, but insertion of the entire VP5 leader and cap site into a construct in which the reporter gene was controlled by the kinetically early (beta) dUTPase (UL50) promoter did not result in any significant change in the kinetics of dUTPase promoter expression. These results suggest that DNA sequences both 5' and 3' of the TATA box are important determinants of the beta gamma kinetics and levels of VP5 mRNA accumulation in the infected cell.

Animals↗

Cloning of the rat insulin-like growth factor-binding protein-2 gene and identification of a functional promoter lacking a TATA box.

We have isolated clones encoding the rat insulin-like growth factor-binding protein-2 (IGFBP-2) gene and determined its organization and nucleotide sequence. The rat IGFBP-2 gene spans at least 8 kilobases and consists of four exons, each of which contains protein-coding sequences. The amino acid sequences of exons 1, 3, and 4 are 32-50% identical to the corresponding exons of human IGFBP-1 and IGFBP-3, and 87-91% identical to those of human IGFBP-2. The 18 cysteines in the mature binding proteins are conserved. Exon 2 shows negligible homology. Primer-extended reverse transcription indicated that the 5' end of IGFBP-2 mRNA is 151 nucleotides up-stream from the translation start site [designated nucleotide (nt) -151]. Consistent with this result, IGFBP-2 mRNA protected a genomic fragment terminating at approximately nt -148, as well as smaller fragments. A 1260 nt fragment containing 1144 nt of 5' flanking region had promoter activity when inserted in the correct orientation into a plasmid containing a promoterless luciferase reporter gene and transiently transfected into BRL-3A rat liver cells, which express IGFBP-2, but not when transfected into H4-II-E cells, which do not express IGFBP-2. The IGFBP-2 gene lacks a TATA box immediately up-stream from the transcription initiation site. It is GC rich (66% between nt -270 and +385) and contains GC boxes that might be recognized by transcription factors Sp1 or ETF. The promoter region contains multiple direct and indirect repeats. One direct repeat contains a variant Sp1 site (-158 to -150) near the consensus Sp1 site at nt -138 to -130. The 5' flanking region also contains motifs that might be recognized by transcription factors AP-1 (Jun/Fos), AP-2, and liver factor B1. The role of these sites in basal and regulated expression of the IGFBP-2 gene remains to be determined.

Amino Acid Sequence↗

Inhibition of basal transcription by poliovirus: a virus- encoded protease (3Cpro) inhibits formation of TBP-TATA box complex in vitro.

Host cell RNA polymerase II (pol II)-mediated transcription is inhibited by poliovirus infection. We demonstrate here that both TATA- and initiator-mediated basal transcription is inhibited in extracts prepared from poliovirus-infected HeLa cells. This inhibition can be reproduced by incubation of uninfected HeLa cell extracts with purified, recombinant poliovirus protease, 3Cpro. Transient-transfection assays demonstrate that 3Cpro, in the absence of other viral proteins, is able to inhibit cellular pol II-mediated transcription in vivo. Three lines of evidence suggest that inactivation of TATA-binding protein (TBP) is the major cause of inhibition of basal transcription by poliovirus. First, RNA pol II transcription in poliovirus-infected cell extract is fully restored by bacterially expressed TBP. Second, addition of purified TBP restores transcription in heat-treated nuclear extracts from mock- and virus-infected cells to identical levels. Finally, using a gel mobility shift assay, we demonstrate that incubation of TBP with the viral protease (3Cpro) inhibits its ability to bind TATA sequence in vitro. These results suggest that inhibition of pol II transcription in mammalian cells infected with poliovirus is, at least in part, due to the inability of modified TBP to bind pol II promoter sequences.

3C Viral Proteases↗

Analysis of structure-function relationships of yeast TATA box binding factor TFIID.

A systematic series of N-terminal, C-terminal, and internal deletion mutants of S. cerevisiae TFIID were expressed in vitro and tested for TATA box binding and basal level transcription activities using, respectively, DNA mobility shift and in vitro transcription assays. The domains responsible for these activities were colocalized to a surprisingly large region containing C-terminal residues 63-240. This region was noted previously to contain potentially interesting structural motifs (central basic core, direct repeats, and sigma factor homology) and, more recently, to be highly conserved among TFIID from different species. Deletion mutant cotranslation studies revealed that TFIID binds DNA as a monomer. The implications of these results for TFIID structure and function are discussed.

Base Sequence↗

Analysis of expression of yeast enolase 1 gene containing a longer pyrimidine-rich region located between the TATA box and transcription start site.

Yeast ENO1 promoter was prepared by a chemical synthetic method. Two variant promoters containing a pyrimidine-rich region (CT block), located between the TATA box and the transcription start site, either 32 or 34 base pairs (bp) longer than the native ENO1 promoter were isolated during the chemical synthesis. Gene expression of variant promoters was compared with that of the native promoter by measuring the amount of mRNA and the activity of beta-galactosidase by constructing ENO1-lacZ gene fusions. No significant differences were observed between the native and variant promoters in transcription levels. The start site of transcription was mapped on CAAG, a consensus sequence of transcription start site of yeast glycolytic genes. The results suggest a longer CT block in ENO1 promoter may not affect the expression of the yeast ENO1 gene. In addition, the level of ENO1 gene expression was found to be higher in stationary phase cells than in log phase cells.

Base Sequence↗

Identification of a vitamin D3-response element that overlaps a unique inverted TATA box in the rat bone sialoprotein gene.

Bone sialoprotein (BSP), an early marker of osteoblast differentiation, has been implicated in the nucleation of hydroxyapatite during bone formation de novo. Our studies, using the osteoblastic cell line ROS 17/2.8, have revealed that rat BSP gene expression is suppressed by 1,25-dihydroxyvitamin D3[1,25(OH)2D3], which is a powerful regulator of bone formation and resorption. To determine the molecular basis of the transcriptional suppression of BSP gene transcription by 1,25(OH)2D3, we have conducted transient transfection analyses with chimaeric constructs of the rat BSP gene promoter linked to a luciferase reporter gene. 1,25(OH)2D3 suppressed expression in all constructs, including a short construct (pLUC 3; nt -116 to +60) that contained a putative vitamin D3-response element (VDRE; AGGGTTTATAGGTCA; nt -28 to -14) that overlaps a unique inverted TATA (TTTATA) box. Mobility shift assays demonstrated strong binding of recombinant human vitamin D3 receptor protein (hVDR) to the VDRE. Point mutations introduced into each half-site and analysed for 1,25(OH)2D3-mediated suppression of transcription and for hVDR binding either decreased or increased both transcriptional suppression and binding. In comparison with activating VDREs, the rat BSP VDRE bound VDR-VDR homodimers more avidly than VDR-RXR alpha heterodimers (where RXR is retinoid X receptor). These studies have therefore identified a novel 1,25(OH)2D3 suppressor element that overlaps the inverted TATA box in the rat BSP gene and indicate that transcriptional suppression of the rat BSP gene by 1,25(OH)2D3 might involve competition between the VDR and the TATA binding protein (TBP).

Animals↗

The same "TATA" box beta-thalassemia mutation in Chinese and US blacks: another example of independent origins of mutation.

A Chinese beta +-thalassemia gene in a new haplotype was chosen for cloning and sequencing. The mutation identified was an A-G transition at position -29 in the TATA box of the beta-globin gene. This mutation has not been seen previously in Chinese but has been documented in American blacks on a different chromosomal background. This observation provides further evidence for independent origins of the same mutation in distinct ethnic groups.

Asian People↗

Promotion of specific in vitro transcription by excised "TATA" box sequences inserted in a foreign nucleotide environment.

We have cloned into plasmid pBR322 a DNA fragment extending from position -32 to position -12 of the adenovirus type 2 major late promoter region (position +1 referring to the cap site). In vitro transcription experiments show that this 21 base pair sequence, which contains the Goldberg-Hogness or "TATA" box, is both necessary and sufficient for specific initiation of transcription by RNA polymerase B (or II). Furthermore, we show that similar sequences, randomly occurring in the bacterial plasmid pBR322, are also recognized by the RNA polymerase B transcription machinery and able to promote specific in vitro transcription. Finally, we discuss the possible importance of the nucleotide sequence of the start region in the actual efficiency of initiation of in vitro transcription.

Adenoviridae↗

A new TATA box mutation detected at prenatal diagnosis for beta-thalassemia.

During the course of prenatal diagnosis for beta-thalassemia in Chinese patients, we encountered a mutation that was not detectable by oligonucleotides for the known Chinese mutations. Amplification of the beta-globin gene by the polymerase chain reaction and direct DNA sequencing revealed a previously undescribed -30 TATA box mutation which was carried by the father. Prenatal diagnosis was achieved, and the fetus did not inherit this beta-thalassemia allele.

Amniocentesis↗

Transcription factor(s) of Ehrlich ascites tumor cells having affinity to the 'TATA' box and a further upstream region of the adenovirus 2 major late gene.

A fraction containing a transcription factor(s) of RNA polymerase II was prepared from a nuclear lysate of Ehrlich ascites tumor cells and its binding to a promoter region of the adenovirus 2 major late gene was examined. Results showed that this fraction contained a factor(s) binding to two distinct regions: a region including the 'TATA' box and another region further upstream. The upstream protected region was different from that reported to be protected by a HeLa cell factor, suggesting species specificity of transcription factor(s) in DNA binding.

Adenoviruses, Human↗

The small nuclear RNA-activating protein 190 Myb DNA binding domain stimulates TATA box-binding protein-TATA box recognition.

Human U6 small nuclear RNA (snRNA) gene transcription by RNA polymerase III requires cooperative promoter binding involving the snRNA-activating protein complex (SNAP(c)) and the TATA-box binding protein (TBP). To investigate the role of SNAP(c) for TBP function at U6 promoters, TBP recruitment assays were performed using full-length TBP and a mini-SNAP(c) containing SNAP43, SNAP50, and a truncated SNAP190. Mini-SNAP(c) efficiently recruits TBP to the U6 TATA box, and two SNAP(c) subunits, SNAP43 and SNAP190, directly interact with the TBP DNA binding domain. Truncated SNAP190 containing only the Myb DNA binding domain is sufficient for TBP recruitment to the TATA box. Therefore, the SNAP190 Myb domain functions both to specifically recognize the proximal sequence element present in the core promoters of human snRNA genes and to stimulate TBP recognition of the neighboring TATA box present in human U6 snRNA promoters. The SNAP190 Myb domain also stimulates complex assembly with TBP and Brf2, a subunit of a snRNA-specific TFIIIB complex. Thus, interactions between the DNA binding domains of SNAP190 and TBP at juxtaposed promoter elements define the assembly of a RNA polymerase III-specific preinitiation complex.

DNA-Binding Proteins↗