No abstract

The 289R EMA protein of adenovirus transactivates a variety of viral and cellular promoters through protein-protein interactions. In earlier studies, mutational analyses of the ElA transactivating domain identified residues that are critical for transactivation and implied that the zinc finger region of the transactivating domain binds a transcription factor. Also, the ElA activation domain was found to bind to the TATA box binding protein (TBP) in vitro. Here, we tested the significance of the E1A-TBP interaction for ElA transactivation by analyzing the effects of conservative substitutions at each of the 49 residues of the ElA activation domain. Seven of the substitutions significantly diminished TBP binding in vitro. All of these were in the zinc finger region and were defective for transactivation in vivo. The perfect correlation between reduced TBP binding and transactivation argues strongly that a direct interaction between the ElA activation domain and TBP is critical to the mechanism of ElA activation. This genetic analysis leads us to further suggest that another factor, which is limiting, is also necessary for ElA-mediated transactivation.The adenovirus ElA 289R protein is a potent transactivator of a variety of viral and cellular promoters (reviewed in refs. 1 and 2). The absence of a common cis element in E1A-inducible promoters (1) and the weak, nonspecific DNA binding activity of the 289R protein (3) suggests that ElA stimulates transcription through protein-protein interactions with cellular transcription factors.The transactivation function of the 289R protein maps to an internal stretch of 49 amino acids (residues 140-188) (4, 5). The salient feature of the ElA transactivating domain is a metal binding structure that is formed by four cysteine residues, which coordinate a single zinc atom (6, 7). The importance of a structurally intact Cys4 zinc finger is highlighted by the fact that replacing any of the four cysteine residues produces a mutant protein that is incapable of activating transcription.Individually substituting every amino acid in the transactivating domain identified additional residues that are also critical for transactivation (8). From this study, ElA proteins containing mutations in a contiguous stretch of residues C terminal to the zinc finger were found not only to be defective for transactivation but also to display a strong transdominant negative phenotype. Furthermore, this study revealed that the transactivating domain is composed of two functionally distinct regions, a finger region (residues 147-177) and a carboxyl region (residues 183-188), each of which is postulated to bind to a different cellular protein. Indeed, two distinct classes of factors, the TATA box binding protein (TBP) and the activating transcription factor family (ATF), have been recently shown to directly interact with the transactivating domain of ElA (9, 10, 34). Taken together, these studies suggest that ElA transactivates by interacting with sequence-specific DNA binding transcription factor...

A 440-base-pair fragment of African green monkey genomic DNA shares homology with the transcriptional regulatory region of simian virus 40 (SV40) and has been reported to direct transcription in vivo. We find that two regions within this fragment bind the promoter-specific cellular transcription factor Spl and are protected in DNase protection ("footprinting") experiments. As in SV40, binding occurs in regions containing multiple copies of the sequence GGGCGG. These regions, when fused to the proximal, or "TATA box," element ofthe herpes simplex virus thymidine kinase promoter, are able to direct Spl-dependent transcription in vitro. The finding that Spl is capable of productive interaction with sequences taken from a cellular promoter supports the idea that Spi may play a role in modulating transcription of cellular genes.Sequences required for the control of transcription and replication in the DNA tumor virus SV40 (simian virus 40) reside in a noncoding region located between divergent transcription units. This 25-base-pair (bp) stretch of DNA contains several regulatory elements that partially overlap but are genetically separable (1). One of these is a bidirectional transcriptional promoter, located in a region containing two exact and one degenerate 21-bp tandem sequence repeats. Nested within these repeats are six copies of the hexanucleotide GGGCGG, known as the "GC box." This promoter element is required for transcription of the early and late viral strands both in vivo and with an in vitro transcription system based on a whole-cell extract (2-11). Previous biochemical studies in our laboratory have identified a cellular factor, Spl, that binds the DNA in the region of the GC boxes and activates transcription by RNA polymerase 11 (12)(13)(14). We have established that Spl is a promoter-specific factor required for SV40 transcription but not for a diverse group of other viral and cellular promoters that were tested.The presence of Spl in uninfected cells strongly suggests that this factor plays a role in cellular as well as viral transcription. The elucidation of this cellular role will be aided by isolation of cellular sequences bearing Spl interaction sites. Several good candidates exist among a group of African green monkey genomic clones (15). These DNAs cross-hybridize to a fragment of SV40 DNA containing the origin and transcriptional control elements and appear to be members of an interspersed repetitive sequence family of about 80 members. Three of these cloned fragments have been sequenced and found to contain multiple copies of the GC box, as well as internal direct sequence repeats and homology to the region of the dyad symmetry in the SV40 replication origin (16).One member of the family, called 7.02, has been characterized extensively and appears to contain a transcriptional promoter (17). Nuclease S1 analysis using probes specific for the 7.02 region, which do not cross-hybridize with other members of the family, detects transcripts proceeding outward from the SV40-like region in b...