Minimal sequence and factor requirements for the initiation of transcription from an atypical, TATATAA box-containing housekeeping promoter

Department of Human Genetics, Roswell Park Cancer Institute, New York State Department of Health, Buffalo 14263.
Journal of Biological Chemistry (Impact Factor: 4.57). 12/1990; 265(33):20524-32.
Source: PubMed


We have established the minimal sequence and factor requirements for both constitutive and viral-induced transcription from an atypical, TATATAA box-containing human housekeeping promoter. Utilizing a transient cotransfection protocol, we have found that efficient transactivation of triosephosphate isomerase (TPI) gene transcription by the immediate early proteins of adenovirus and pseudorabies virus is dependent upon the same assembly of sequence elements that collectively confer minimal TPI promoter function in the absence of viral protein. These elements span TPI promoter positions -65 and -6 (where +1 is the transcription initiation site) and include not only a TFIID-responsive TATATAA box (-27 to -21) but a single GC box (-53 to -48) that binds Spl, and a novel cap proximal element (-18 to -6) that binds a 110-kDa nuclear factor that is present in HeLa cells. We demonstrate that these elements function in an interdependent fashion; deleting either GC box 1 or the cap proximal element completely or nearly abolished both basal transcription and viral transactivation. Therefore, these elements and their cognate factors represent the basal transcription initiation complex through which the immediate early protein of adenovirus or pseudorabies virus mediates the stimulation of TPI gene transcription. We discuss the implications of these data for both constitutive and viral-induced transcription.

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    • "The level of TPI activity is increased in dividing cells (Old et al. 1989), and, interestingly, an additional isozyme is detected in rapidly dividing human cells (Decker and Mohrenweiser 1986). Although sequences overlapping and/or immediately 5' of the transcription-initiation site have been found to be essential for transcription of a series of genes, no obvious consensus sequences have been identified (Boyer and Maquat 1990; Weis and Reinberg 1992). Thus, it is not possible to suggest a mechanism by which nucleotide substitutions within the CPE element prevent the binding of the protein identified by Boyer and Maquat (1990), but the data from these studies are consistent with a functional significance for the A-5/G-8 substitutions. "
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    ABSTRACT: Individuals with 50% of expected triosephosphate isomerase (TPI) enzyme activity have been previously identified in families during the screening of approximately 2,000 newborn children for quantitative variation in activity of 12 erythrocyte enzymes. The frequency of the trait was 9/1,713 individuals in the Caucasian population and 7/168 individuals among the African-American population studied. Genetic transmission of the trait was confirmed in all families. The frequency of the presumptive deficiency allele(s) at the TPI locus was greater than expected, given the reported incidence of clinical TPI deficiency. We report the molecular characterization of the variant alleles from seven African-American and three Caucasian individuals in this group of unrelated individuals. Three amino acid substitutions--a Gly-->Ala substitution at residue 72, a Val-->Met at residue 154, and a previously described Glu-->Asp substitution at residue 104--were identified in the Caucasian individuals. The substitutions occur at residues that are not directly involved in the active site but are highly conserved through evolutionary time, suggesting important roles for these residues in maintenance of subunit structure and conformation. The variant allele in the seven African-American individuals had nucleotide changes at positions -8 and -5 (5' of) from the transcription-initiation site. In three of these individuals, an additional T-->G substitution was detected in a TATA box-like sequence located 24 nucleotides 5' of the transcription-initiation site and on the same chromosome as the -5/-8 substitutions. Thus, molecular alterations at the TPI locus were detected in 10 unrelated individuals in whom segregation of a phenotype of reduced TPI activity previously had been identified.
    The American Journal of Human Genetics 02/1996; 58(2):308-16. · 10.93 Impact Factor
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    ABSTRACT: We identify DNA regions that are necessaryfortheubiquitous expression oftheDrosophila melanogaster oaI-tubulin (ailt) gene.Invitro transcription showedthattwoupstreamregions, tubulin element 1(TE1(29 bp))andtubulin element 2 (TE2(68bp)), anda downstream region activate transcription. Germline transformation demonstrated thatthese three regions aresufficient todirect theotlt corepromoter tobegin transcribing atthestageofcellular blastoderm formation andtocontinue thereafter athighlevels inalltissues anddevelopmental stages. Remarkably, mutation ofany one oftheseregions results inhighsensitivity to chromosomal position effects, producing different butreproducible tissue-specific patterns ofexpressionin eachtransformed line. Noneoftheseregions behaves as an enhancer ina conventional germ line transformation test. Theseobservations showthatthesethreeregions, twoofwhichbindtheGAGA transcription factor, actubiquitously toinsulate fromposition effects andtoactivate transcription. Theresults alsoprovide vectors forubiquitous expression ofgeneproducts andforexamining silencer activities. Cellviability depends ona large numberofubiquitous proteins, suchastubulins, histones, andenzymes, involved in basic cellular metabolism. Thisraises thequestion ofhow genesencoding theseubiquitous proteins areregulated in multicellular organisms whosecells undergo dramatic changes ingeneregulation during differentiation. Expression ofubiq- uitous genesmaybeentirely independent oftheregulatory programs that direct differential geneexpression. Forexample, expression couldresult solely fromtheaffinity between the corepromoter andgeneral transcription factors present inall cell types. Alternatively, ubiquitous transcription ofagenemay bedirected bythese regulatory programs. Forexample, expres- sioncouldresult fromtheactivity ofalarge collection of cell-type-specific enhancers. Inlight ofthese widely different possibilities, itissurprising that relatively little attention hasbeenfocused ontheregula- tion ofubiquitously expressed genes. Theinvestigations that havebeenconducted suggest that, ingeneral, suchgenesare expressed atlowlevels, lackaTATA promoter element, and contain GC-rich regions upstream ofthetranscription initia- tion site (11). However, there arealmost asmanyexceptions to these generalizations asthere areagreements withthem. For example, themousePgk-1 promoter isexpressed athighlevels, thehumantriosephosphate isomerase genecontains aTATA element, andtheDrosophila melanogaster otl-tubulin (otlt) genehasaTATA element andlacks GC-rich regions (3,36, 51). Furthermore, understanding ofubiquitous regulation has beenlimited because invivoanalyses havebeendonewithcell culture assays whichdonottest thefull rangeofpotential tissue specificities oftheDNA elements involved (for example, seereferences 3,6,and36). Thisreport examines DNA regions thatregulate theDro-
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    ABSTRACT: Regulation of eukaryotic messenger RNA transcription is governed by DNA sequence elements that serve as binding sites for sequence-specific transcription factors. These include upstream and downstream promoter-proximal elements, enhancers, repressors, and silencers, which modulate the rate of specific initiation by RNA polymerase II. In addition, the promoter-proximal region between -45 and +30 (relative to the start of initiation) contains two highly conserved motifs, the TATA sequence at around -30 and CA at +1. Although the TATA element-binding factor TFIID has been purified and cloned from several organisms and has provided invaluable insight into the process of transcription initiation and its regulation, little is known about factors that interact at the +1 region. We have recently shown that the adeno-associated virus type 2 P5 promoter +1 region (P5 + 1 element) binds transcription factor YY1. We report here that this sequence is necessary and sufficient for accurate basal transcription. Further, partially purified YY1 can restore basal level transcription from a P5 + 1 element in a HeLa extract depleted for YY1 or a Drosophila embryo extract devoid of YY1 activity, whereas a YY1-specific antibody can block the reactivation. Finally, using electrophoretic mobility shift assay, we have identified YY1-related factors that bind to two other transcription initiators in cellular genes.
    Nature 12/1991; 354(6350):241-5. DOI:10.1038/354241a0 · 41.46 Impact Factor
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