Purification andfunctional characterization ofacellular transcription factor thatbinds toanenhancer element within theadenovirus early Ellapromoter
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ABSTRACT The adenovirus Ela-inducible early ElIa(EIIaE) promoter is comprised of several sequence elementsessential for constitutive and induced expression. Wereportherethe purification ofthe host-cell factor that interacts withthe major upstream element of this promoter, extendingbetween positions -90 and -70 with respect to the mainEIaEcapsite andexhibiting enhancer properties. Thepuri-fied factor, whichcorrespondstoa40-to43-kDapolypeptide,specifically binds to its recognition site and stimulates EIIaEpromoter activity when added to an in vitro transcriptionsystem,reconstitutedfrompurifiedfactorsandRNApolymer- ase. Theimplication of this factor in the control ofthe otheradenovirusearly genes is discussed.Efficient transcription of the adenovirus early transcriptionunits requires the presence of the viral pre-early EIageneproducts(1, 2). Themechanismofthis transactivation oftheearly transcription unit is still poorlyunderstood. Extensivedeletion andlinkerscanningmutational analysis (3-5) oftheEIa-inducibleEllaearly(EIlaE)promoterhasindicatedthatKeywords:
Transcription
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Transcription
TAF2
Response element
General transcription factor
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The sequence requirements for transcriptional stimulation of the adenovirus major late promoter (MLP) by the products of the early transcription unit Ela and by the replication of viral DNA were analyzed by in vitro transcription. Sequences upstream of +33 are involved in the moderate Ela-responsiveness of the MLP, while sequences between +33 and +131 are required for its major replication-induced transcriptional activation. Dnase I footprinting experiments delineate a sequence component, extending from +76 to +120, which binds protein(s) only in extracts of cells where viral DNA replication occurred. Taken together, these results suggest that the replication-dependent stimulation of the MLP is mediated by the increased binding of this protein(s).
Transcription
Replication factor C
Origin recognition complex
Licensing factor
Footprinting
DNA footprinting
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The tegument proteins of human cytomegalovirus are introduced into cells as components of infectious virus. The tegument proteins may affect viral and cellular transcription prior to the synthesis of the immediate-early viral regulatory proteins. The phosphorylated tegument protein of 71 kDa (pp71) is reported to be encoded by the UL82 gene. The UL82 gene products transactivated promoters containing upstream ATF or AP-1 binding sites. In contrast, the phosphorylated tegument protein of 65 kDa (pp65), encoded by the UL83 gene, had no detectable effect on these promoters. Enhancement by UL82 of downstream transcription was directly proportional to the number of upstream ATF sites. Response to UL82 transactivation was abolished by mutation of the ATF site. Mutation in the carboxy-terminal region of UL82 also eliminated transactivation. Even though the major immediate-early promoter of human cytomegalovirus is a strong enhancer-containing promoter, UL82 further enhanced its transcription as much as 20-fold. The mechanism of UL82 enhancement of transcription from viral or cellular promoters is not known, but the enhancement may be mediated by triggering one of the protein kinase signaling pathways, increasing the affinity of ATF or AP-1 for the target sequence, or stabilizing the complex between the eucaryotic transcription factor and the target sequence.
Viral tegument
Upstream activating sequence
Transcription
TATA box
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cis-Acting elements involved in transcription of the peptide IX (pIX) gene of adenovirus 2 were identified by using in vivo transient expression assays and two in vitro transcription systems. Deletion of either the sequence between positions -45 and -70 or the TATA box abolished the initiation of pIX gene transcription in vivo and transcription with HeLa cell nuclear extracts in vitro. These results initially suggested the presence of a positive factor acting on the upstream element. However, when proteins in the nuclear extract were fractionated by column chromatography and analyzed by reconstitution of transcription in vitro, it was found that a certain fraction could direct TATA box-dependent transcription initiation even in the absence of the upstream element. Furthermore, activity inhibiting TATA box-dependent transcription was found in the nuclear extract. In contrast, inhibition of TATA box-dependent transcription was suppressed by deletion of a downstream sequence between positions +33 and +122. These results indicate that the TATA box of the pIX gene by itself has the ability to direct initiation of constitutive transcription but that the function of this element is under negative control by a repressor acting on a downstream sequence. Thus, the upstream element of the pIX gene appears to have a novel function: suppression of the transcriptional repression exerted by a downstream sequence, leading to a net transcription activation. Possible mechanisms for transcription initiation of pIX DNA are discussed.
TATA box
Transcription
Upstream activating sequence
E-box
Response element
General transcription factor
CAAT box
TAF2
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Sequence (biology)
Transcription
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TATA box
TAF1
TAF2
General transcription factor
Upstream activating sequence
Transcription preinitiation complex
TATA-Box Binding Protein
Transcription
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We have examined the relationship between sequence-specific DNA-binding proteins that activate transcription of E1A-inducible adenovirus early promoters. Factors previously referred to as E4F1 and E2A-EF bind to the E4 and E2A promoters, respectively. We demonstrate here that E4F1 and E2A-EF have identical DNA-binding specificity. Moreover, E4F1 and E2A-EF both activate transcription of the E4 and E2A promoters in vitro. These findings demonstrate that E4F1 and E2A-EF are the same factor, which we have designated activating transcription factor, or ATF. In addition to the E4 and E2A promoters, ATF binds to an important functional element of the E1A-inducible E3 promoter. Interaction of a common activator protein, ATF, with multiple E1A-inducible early viral promoters, suggests a significant role for ATF in E1A-mediated transcriptional activation.
Response element
General transcription factor
Sp1 transcription factor
Transcription
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TAF1
TATA box
TAF2
General transcription factor
TAF4
TATA-Box Binding Protein
Transcription
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The circular polyomavirus genome is transcribed from divergent promoter regions. Early mRNAs are initiated from a transcription complex formed at a TATA motif, the site of binding of transcription factor TFIID. Early transcription is promoted at a distance by the viral enhancer, which includes DNA motifs bound by cellular proteins of the PEA1 and PEA3 families of transcription activators. In contrast, the predominant viral late mRNAs are initiated within the viral enhancer, which lacks a TATA motif, near the PEA1 and PEA3 DNA motifs. Here, we demonstrate that these PEA1 and PEA3 binding sites are primary components of an autonomous transcription initiator element (Inr). They cause transcription of most polyomavirus late mRNAs and can direct the transcription of heterologous reporter genes. Alternative roles of these DNA motifs as activators of early mRNA transcription and as an initiator element for late mRNA transcription help explain how polyomavirus gene expression is regulated during lytic growth and provides a model for cellular transcription during development.
E-box
TAF2
Transcription
General transcription factor
TATA box
Response element
RNA polymerase II
Enhancer RNAs
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Transcription preinitiation complex
Transcription
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Citations (49)