AR1 IS AN INTEGRAL-PART OF THE ADENOVIRUS TYPE-2 E1A-CR3 TRANSACTIVATION DOMAIN

We have previously shown that the nonconserved carboxy-terminal exon o f the adenovirus type 2 E1A-289R protein contains two interchangeable sequence elements, auxiliary region (AR) 1 and AR2, that are required for efficient CR3-mediated transcriptional activation of the viral E4 promoter (M. Bondesson, C. Svensson, S. Linder, and G. Akusjarvi, UMBO J. 11:3347-3354, 1992). Here we show that CR3-mediated transactivatio n of all adenovirus early promoters and the HSP70 promoter requires th e AR1 element, We further show that AR2 can substitute for AR1 only wh en artificially juxtaposed to CR3, AR1 consists of six tandem glutamic acid-proline (EP) repeats and is positioned immediately downstream of CR3, Genetic dissection of AR1 showed that the number of EP repeats i n AR1 is critical for CR3 function. Thus, reducing or increasing the n umber of EP repeats reduces the CR3 transactivation capacity. Furtherm ore, the introduction of amino acid substitutions into AR1 suggested t hat the net negative charge in AR1 is of critical importance for its f unction as an enhancer of CR3-mediated transcriptional activation. Usi ng an in vitro binding approach, we showed that the AR1 element is not part of the CR3 promoter localization signal mediating contact with t he Spl, ATF-2, or c-Jun upstream-binding transcription factors. Previo us studies have suggested that the 49-amino-acid sequence constituting CR3 represents the minimal domain required for E1A-induced activation of viral early promoters. Since AR1 was required for efficient CR3-me diated transcriptional activation of all tested promoters, we suggest that the carboxy-terminal boundary for the CR3 transactivation domain should be extended to include the AR1 element.