ICP4, the major transcriptional regulatory protein of herpes simplex virus type 1, forms a tripartite complex with TATA-binding protein and TFIIB.
The ICP4 protein of herpes simplex virus can either increase or decrease the rate of transcription mediated by RNA polymerase II, depending on the target promoter. The interplay of DNA-protein and protein-protein contacts determining ICP4 function has yet to be characterized, and consequently the molecular mechanism by which the protein acts remains unclear. ICP4 can transactivate minimal promoters containing only TATA homologies, and therefore it is reasonable to hypothesize that ICP4 works by influencing the TATA-dependent assembly of general transcription factors via specific protein-protein interactions. This study directly addresses this hypothesis by determining whether ICP4 affects the assembly of general transcription factors on templates bearing a TATA box and an ICP4-binding site. Using gel retardation and footprinting assays, we found that ICP4 forms a tripartite complex with TFIIB and either the TATA-binding protein (TBP) or TFIID. The formation of this complex was not the result of simple tripartite occupancy of the DNA but the consequence of protein-protein interactions. In the presence of all three proteins, the affinity of ICP4 and TBP for their respective binding sites was substantially increased. Using mutant derivatives of ICP4 and defective versions of promoters, we also demonstrated that the ability of ICP4 to regulate gene expression correlated with its ability to form a tripartite complex with TFIIB and TBP in vitro.
Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pennsylvania 15261.
C A
CA
Smith
P
P
Bates
R
R
Rivera-Gonzalez
B
B
Gu
N A
NA
DeLuca
D000818:Animals; D001665:Binding Sites; D004268:DNA-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D003850:Deoxyribonuclease I; D015967:Gene Expression Regulation, Viral; D017874:Immediate-Early Proteins; D008961:Models, Structural; D016296:Mutagenesis; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid; D018139:Simplexvirus / Q000235:genetics / Q000378:metabolism*; D016385:TATA Box; D035181:TATA-Box Binding Protein; D035581:Transcription Factor TFIIB; D014157:Transcription Factors / Q000302:isolation & purification / Q000378:metabolism*; D014158:Transcription, Genetic; D014709:Vero Cells; D054334:Viral Regulatory and Accessory Proteins / Q000302:isolation & purification / Q000378:metabolism*
D004268:DNA-Binding Proteins; D017874:Immediate-Early Proteins; D011994:Recombinant Proteins; D035181:TATA-Box Binding Protein; D035581:Transcription Factor TFIIB; D014157:Transcription Factors; D054334:Viral Regulatory and Accessory Proteins; C048925:herpes simplex virus, type 1 protein ICP4; D003850:Deoxyribonuclease I