Adenovirus EIIA early promoter: transcriptional control elements and induction by the viral pre-early EIA gene, which appears to be sequence independent.

Abstract

A molecular dissection of the adenovirus EIIA early (E) promoter was undertaken to study the sequence elements required for transcription and to examine the nucleotide sequences, if any, specific for its trans-activation by the viral pre-early EIA gene product. A chimeric gene in which the EIIA-E promoter region fused to the coding sequences of the bacterial chloramphenicol acetyltransferase (CAT) gene was used in transient assays to, identify the transcriptional control regions. Deletion mapping studies revealed that the upstream DNA sequences up to -86 were sufficient for the optimal basal level transcription in HeLa cells and also for the EIA-induced transcription. A series of linker-scanning (LS) mutants were constructed to precisely identify the nucleotide sequences that control transcription. Analysis of these LS mutants allowed us to identify two regions of the promoter that are critical for the EIIA-E transcription. These regions are located between -29 and -21 (region I) and between -82 and -66 (region II). Mutations in region I affected initiation and appeared functionally similar to the "TATA" sequence of the commonly studied promoters. To examine whether or not the EIIA-E promoter contained DNA sequences specific for the trans-activation by the EIA, the LS mutants were analyzed in a cotransfection assay containing a plasmid carrying the EIA gene. CAT activity of all of the LS mutants was induced by the EIA gene in this assay, suggesting that the induction of transcription of the EIIA-E promoter by the EIA gene is not sequence-specific.By using a variety of assays, the essential nucleotide sequences and functional domains of a number of cellular and viral RNA polymerase II promoters have been identified. The majority of these promoters contain two essential control elements. The first such element, the "TATA" box located between 20 and 30 base pairs (bp) upstream from the initiation site, positions the initiation of transcription (1, 2). Another type of element that is usually found between 50 and 150 bp from the initiation site is required for efficient transcription (3)(4)(5)(6)(7)(8)(9)(10)(11)(12) (21). Both of these promoters are somewhat unusual in that they lack TATA box elements (21). At present it is not clear whether these promoters lacking the TATA box utilize in its place an analogous sequence element or whether they belong to a separate class of polymerase II promoters that are differently regulated. The EIIA-E promoter that functions early in infection is also subject to positive regulation. A 289 amino acid phosphoprotein encoded by the pre-early EIA region is required for the activation of the EIIA-E promoter and other early viral promoters following the Ad infection of permissive human cells (22)(23)(24).A detailed mutational analysis of the EIIA-E promoter was undertaken in this investigation to identify the DNA sequences required for transcription and to determine whether or not this promoter contains nucleotide sequences or domains that are responsible for its trans-activat...

Trans-activation of the adenovirus E2 promoter by human papillomavirus type 16 E7 is mediated by retinoblastoma-dependent and -independent pathways

“…This latter possibility would explain the reduction in the level of trans-activation of the E2 promoter when elements which alone are insufficient to mediate E7 trans-activation are deleted (Phelps et al, 1991). Two modified reporter plasmids have been analysed to determine these possibilities: the construct LS-74/85 contains a BamHI linker insertion within the activating transcription factor (ATF) site (Murthy et al, 1985) and in A74-CAT this site has been used to remove all E2-derived sequences upstream of the distal ATF site by digesting with XhoI plus BamHI, infilling then recircularization. Mutants lacking the E2F sites were not analysed as these were assumed to be deficient for both pathways of E7 trans-activation, based on the work of Phelps et al (1991).…”

“…This yielded an insert in the pJ4f2 vector consisting of the E 7 0 R F preceded by a C residue derived from the initial cloning. The E7-responsive reporter plasmid E2-CAT, which contains the adenovirus E2 early promoter directing transcription of the chloramphenicol acetyltransferase (CAT) gene, has been previously described (Murthy et al, 1985). A flgalactosidase-expressing internal control plasmid was constructed by transferring the fl-galactosidase coding region from pJ6-flgal (gift of Dr N. Jones) to pJ4~ using the HindIII and BglII restriction sites.…”

Some Highlights of Animal Virus Research in 1985

“…The mechanisms of these activation effects are generally obscure and presumably diverse (Alwine, 1985). It is likely that both E 1 a and IE 175 act in a manner independent of promoter sequence (Everett, 1984 b;Murthy et al, 1985;Left et al, 1985).…”

The HPV-16 E7 oncoprotein binds Skip and suppresses its transcriptional activity

“…The cellular Ski expression plasmid, pMT2-Ski, was kindly provided by MJ Hayman (Dahl et al, 1998). CAT reporter constructs pBLCAT2 and AdE2CAT have been described previously (Luckow and Schutz, 1987;Murthy et al, 1985). For making His 6 -16 E7, the gene was cloned in pET7His6 at BamHI-EcoRI sites.…”