Abortive initiation and first bond formation at an activated adenovirus E4 promoter

Abortive initiation at the adenovirus E4 promoter was studied by following the production of RNA formed from the initiating nucleotides UpA and CTP. Formation of a specific short RNA via a reaction with appropriate alpha-amanitin sensitivity required promoter, activator, and ATP. In the absence of any of these, an alpha-amanitin-resistant reaction led to lower levels of a product of unknown origin. The alpha-amanitin-sensitive reaction required open promoter complexes, as assayed directly by permanganate probing. This reaction was not blocked by the inhibition of polymerase C-terminal domain kinase activity or by the lack of DNA supercoiling. Thus, formation of the initial bond of the mRNA appears to require activator and ATP to open the DNA but not phosphorylation of the polymerase C-terminal domain. In addition, the abortive initiation reaction was strongly suppressed when all elongation substrates were present, suggesting that cycling to produce high amounts of abortive product is strongly disfavored during productive initiation at this promoter.