Characterization of the interaction between the acidic activation domain of VP16 and the RNA polymerase II initiation factor TFIIB.
Contact between a transcriptional activator and one or more components of the RNA polymerase II transcription initiation machinery is generally believed important for activators to function. Several different molecular targets have been suggested for direct contact by herpes simplex virus virion protein VP16, including the general initiation factor TFIIB. In this report we have used several strategies to critically assess this interaction between VP16 and TFIIB. Affinity columns of VP16 bound TFIIB activity from HeLa cell extracts and the binding was reduced by mutations in the activation domain of VP16. In assays of direct binding, VP16 bound recombinant human TFIIB but not Drosophila or yeast TFIIB. Unlike binding from an extract, however, we found that the interaction between VP16 and recombinant human TFIIB was not affected by mutations in VP16 that reduce transactivation. Point mutations within human TFIIB that reduce transactivation by VP16 have been shown to reduce VP16 binding, but we show here that these same mutations critically affect both the important TBP-TFIIB interaction and the ability of TFIIB to support activator-independent basal transcription in vitro. Taken together our results suggest more evidence is needed to support the notion that TFIIB is a functionally important target for the activator VP16.
Gupta
R
R
Banting and Best Department of Medical Research, University of Toronto, Ontario, Canada.
Emili
A
A
Pan
G
G
Xiao
H
H
Shales
M
M
Greenblatt
J
J
Ingles
C J
CJ
D000818:Animals; D001665:Binding Sites; D002846:Chromatography, Affinity; D004330:Drosophila / Q000378:metabolism; D005656:Fungal Proteins / Q000378:metabolism; D006367:HeLa Cells; D017405:Herpes Simplex Virus Protein Vmw65 / Q000378:metabolism*; D018259:Herpesvirus 1, Human / Q000378:metabolism*; D006801:Humans; D009154:Mutation; D011485:Protein Binding; D012319:RNA Polymerase II / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D015534:Trans-Activators / Q000378:metabolism; D035581:Transcription Factor TFIIB; D035362:Transcription Factor TFIID; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
D005656:Fungal Proteins; C070633:Gal-VP16; D017405:Herpes Simplex Virus Protein Vmw65; D011993:Recombinant Fusion Proteins; D015534:Trans-Activators; D035581:Transcription Factor TFIIB; D035362:Transcription Factor TFIID; D014157:Transcription Factors; D012319:RNA Polymerase II