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Alpha-Amanitin is a well-known specific inhibitor of RNA polymerase II (RNAPII) in vitro and in vivo. It is a cyclic octapeptide which binds with high affinity to the largest subunit of RNAPII, RPB1. We have found that in murine fibroblasts exposure to alpha-amanitin triggered degradation of the RPB1 subunit, while other RNAPII subunits, RPB5 and RPB8, remained almost unaffected. Transcriptional inhibition in alpha-amanitin-treated cells was slow and closely followed the disappearance of RPB1. The degradation rate of RPB1 was alpha-amanitin dose dependent and was not a consequence of transcriptional arrest. Alpha-Amanitin-promoted degradation of RPB1 was prevented in cells exposed to actinomycin D, another transcriptional inhibitor. Epitope-tagged recombinant human RPB1 subunits were expressed in mouse fibroblasts. In cells exposed to alpha-amanitin the wild-type recombinant subunit was degraded like the endogenous protein, but a mutated alpha-amanitin-resistant subunit remained unaffected. Hence, alpha-amanitin did not activate a proteolytic system, but instead its binding to mRPB1 likely represented a signal for degradation. Thus, in contrast to other inhibitors, such as actinomycin D or 5,6-dichloro-1-beta-D-ribofuranosyl-benzimidazole, which reversibly act on transcription, inhibition by alpha-amanitin cannot be but an irreversible process because of the destruction of RNAPII.

The cDNA of the second largest subunit of RNA polymerase II (or B) from HeLa cells has been cloned and sequenced. A predicted amino acid sequence of 1174 residues (calculated molecular mass of 133,896 Da) was derived from the longest open reading frame and compared to the sequences of homologous subunits of polymerases of eukaryotic, archaeal and bacterial origin. After optimal alignment, about 16% of the residues were found to be conserved throughout evolution, from human to Escherichia coli. About 2/3 of the overall length of the conserved domains delineated by these residues are clustered within the C-terminal half of the human polypeptide, whereas the remaining is spread over its N-terminal half. The putative functional significance of these conserved domains is discussed.

Transcription from the adenovirus major late promoter (MLP) is greatly stimulated during lytic infection, after replication of the viral DNA has started. This replication-dependent activation has previously been shown to be mediated by a positive regulatory cellular protein(s). Binding of this factor(s) to sequence elements (DE1 and DE2), located between positions +76 and +124, with respect to the MLP transcriptional startsite, is detected only after the onset of DNA replication. Using a cell-free transcription system which mimics the late phase induction of the MLP and DNA binding assays, we now present evidence showing that maximal stimulation also depends on the MLP upstream element (UE), without involving increased DNA binding activity of the corresponding factor (UEF) during the lytic cycle. Our results indicate that the upstream and downstream elements act cooperatively on transcription efficiency, although no direct interactions between the cognate factors could be demonstrated. These observations strongly suggest that the elevated rate of transcription originating at the MLP startsite, late in infection, results from the simultaneous action of factors bound at the upstream and downstream elements onto a common target within the basal transcription machinery.

The murine embryonal carcinoma (EC) F9 cells express an adenovirus EIa-like activity which is abolished after differentiation of these cells. We have examined the ability of the adenovirus EIa gene products to transactivate the viral early EIIa (EIIaE) promoter in this cell system. Surprisingly, as shown both by infection and transfection experiments, the EIIaE promoter was refractory to viral EIa-mediated activation in the undifferentiated F9 EC cells, EIa-responsiveness being recovered only after differentiation of these cells. This EC-cell-specific restriction did not correspond to a lack of EIa gene expression, nor to a deficiency of any of the major transcription factors involved in EIIaE basal promoter activity. In these differentiated F9 cells, EIIaE promoter activation correlates with a modification of E2F resulting in its ability to cooperatively interact with the promoter. Our observation that such a modification neither occurs in uninfected F9 EC cells (Jansen-Durr et al., 1989, EMBO J., 8, 3365), nor at early times after infection of these cells suggests that a viral product is required together with the EIa-like activity to induce this modification of E2F. The implication of an EIV gene product in this effect is discussed.

A series of deletion mutants extending from -250 toward the capsite has been constructed in the early promoter region of the adenovirus 2 EIIa gene and tested both in vitro, and in vivo after transfection of HeLa cells, for the ability to act as a template for transcription. A region between positions -94 and -63 upstream from the major EIIa early cap site is essential both in vivo and in vitro for efficient promoter function. By cotransfection of the EIIa deletion mutants with the EIa transcription unit it has been possible to demonstrate that deletion to position -94 does not affect induction of transcription of the EIIa early gene by the EIa transcription unit, but deletion to position -63 results in loss of detectable levels of EIIa early specific RNA. Thus, sequences upstream from position -94 of the EIIa early gene are not involved in the induction of the EIIa early gene by the EIa transcription unit.

By transient expression analysis in HeLa cells of adenovirus-2 E2a early (E2aE) promoter mutants and hybrid E2aE-beta-globin gene constructs, we demonstrate the existence of three nucleotide (nt) sequence elements involved in the E1a-responsiveness of the E2aE transcription unit: element I, localized within a segment (nt -13 to +62) surrounding the major E2aE cap site (nt + 1); element II (between nt -71 and -29), and element III (between nt -146 and -86). Each element is unable by itself to confer E1a responsiveness. Only combinations of sequences including elements I and II (spanning nt -71 and +62) or II and III (spanning nt -146 and -29) ensure maximal inducibility, for which element II appears of central importance.

As an approach to the identification of essential factors required for specific expression of the adenovirus type 2 EIIaE early (EIIaE) promoter, an in vitro system was established. Under appropriate conditions, using crude extracts of non-infected HeLa cells, efficient and accurate EIIaE expression has been reproduced. As in vivo, this transcription was strongly dependent upon the integrity of two non-consensus TATA-like elements, T1 and T2, corresponding to the major (EIIaE1) and minor (EIIaE2) start sites, respectively, as well as upon intact upstream elements (A, between -40 and -50 and B, between -70 and -90) common to both overlapping promoters. The implication of specific DNA-binding proteins in the transcriptional effects mediated by these elements was demonstrated by DNAse I footprinting analyses. Both crude nuclear extracts and partially purified fractions confer specific protection against DNAse I digestion to the T1 and B promoter elements defined above, and to a far upstream region (element C, between -110 and -150), which has previously been identified as a weaker promoter element by in vivo transcriptional studies. Separation of the T1 recognition factor from those which bind to the upstream elements B and C by chromatographic fractionation of the extracts has also been achieved.