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...
The transcriptional control elements of the Adenovirus (Ad) type 5 EIIA‐late (L) promoter were analyzed in the context of the viral chromosome. Promoter mutants constructed in vitro [deletion and linker‐scanning (LS)] were re‐introduced into the non‐essential EIII region of an Ad5 variant which lacked the EIA gene. They were then analyzed in human 293 cells for EIA‐dependent and in HeLa cells for EIA‐independent transcription. These studies revealed that a minimum of approximately 157 bp upstream from the Cap site are sufficient for the efficient transcription of this promoter in the presence or absence of the EIA gene products. Within the 157‐bp sequence, multiple control elements can be identified. These are (i) a sequence block between −55 and −21 which contained a sequence resembling the TATA box and an Sp1 recognition site 5′‐TGGGCGTGGT‐3′, (ii) a sequence block between −84 and −67 which contained a second Sp1 recognition sequence, 5′‐CGGGCGGGAT‐3′ and a 5′‐CCAAT‐3′ box in the non‐coding strand and (iii) a 56‐bp sequence block between −157 and −101 which contained a 5′‐CCAAT‐3′ sequence in the non‐coding strand. The transcriptional pattern of the LS mutants in 293 cells was very similar to that of HeLa cells suggesting that neither of the EIA gene products interact with EIIA‐L promoter directly to modulate transcription. A purified Sp1 protein protected DNA sequences from −56 to −33 which includes the Sp1 recognition sequence closer to the cap site whereas the distal Sp1 recognition sequence showed a very weak affinity for the Sp1 factor.(ABSTRACT TRUNCATED AT 250 WORDS)
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