Pseudorabies virus early protein 0 trans-activates the TATA-associated promoter by stimulating the transcription initiation

Pseudorabies virus (PRV) early protein 0 (EP0) is a transactivator containi ng a RING finger domain. To assess the transactivation mechanism of PRV EP0 , we performed the in vitro transcription by combining HeLa nuclear extract , purified recombinant EP0 and simple promoter constructs, and evaluated th e results by primer extension. The data showed that EP0 could significantly activate the TATA-containing synthetic promoters. Moreover, EP0 activated transcription by stabilizing the formation of transcription initiation comp lex instead of enhancing the elongation rate. To further understand the rol e of EP0 on assembling the transcription initiation complex, we performed t he pull-down assay using affinity precipitation of proteins from HeLa nucle ar extracts and bacterially expressed glutathione-S-transferase EP0 RING fi nger fusion. The data showed that at least six nuclear proteins physically interacted with the EP0 RING finger. Overall, the transactivation of PRV EP 0 is accomplished by enhancing the transcription initiation and is associat ed with at least six nuclear proteins. (C) 1999 Published by Elsevier Scien ce B.V. All rights reserved.