Genetic dissection of the transactivating domain of the E1a 289R protein of adenovirus type 2.
A series of linker-scanning, deletion, and frameshift mutations were made in the pm975 variant of the adenovirus type 2 E1a gene, which expresses only the larger of the two major E1a proteins. Most of these were within the 46-amino-acid segment unique to the larger E1a protein product (the 289R protein), which confers on it the ability to activate in trans the expression of other genes. The mutations were recombined into virus and assayed by in vitro transcription in nuclei isolated from infected cells for their ability to activate the transcription of other viral early genes and of the endogenous hsp70 gene. Mutant E1a proteins from which the 289R-unique segment was removed by deletion or truncation did not completely lose the ability to transactivate by comparison with a virus which makes no E1a at all, indicating that sequences outside this domain are active in the positive regulation of transcription. The E1a mutations tested fell into several classes: those that increased transactivation of virtually all genes, those that severely depressed transactivation of all genes, and those that depressed transactivation only moderately. Each mutation had similar effects on the expression of all transcription units tested, indicating a common process in their transactivation. However, some mutants in the third category decreased transactivation of some induced genes more severely than of others. Such gene-specific defects suggest the existence of subclasses of E1a-responsive transcription units, consistent with the involvement of diverse proteins in the transactivation of different genes. Two specific structural components of the transactivating domain, a putative metal-binding element and a region with high potential for beta-sheet formation at its carboxy-terminus, appear to be important to the transactivation function.
Fahnestock
M L
ML
Fred Hutchinson Cancer Research Center, Seattle, Washington 98104.
Lewis
J B
JB
D017456:Adenovirus Early Proteins; D000260:Adenoviruses, Human / Q000235:genetics*; D000595:Amino Acid Sequence; D004252:DNA Mutational Analysis; D005786:Gene Expression Regulation; D006367:HeLa Cells; D008969:Molecular Sequence Data; D009856:Oncogene Proteins, Viral / Q000235:genetics* / Q000648:ultrastructure; D011487:Protein Conformation; D013329:Structure-Activity Relationship; D014158:Transcription, Genetic
D017456:Adenovirus Early Proteins; D009856:Oncogene Proteins, Viral