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J Biol Chem, Vol. 274, Issue 49, 34819-34824, December 3, 1999

The Use of ATP and Initiating Nucleotides during Postrecruitment Steps at the Activated Adenovirus E4 Promoter

From the Department of Chemistry and Biochemistry, Molecular Biology Institute, UCLA, Los Angeles, California 90095-1569

Permanganate probing has been used to follow the progress and ATP dependence of promoter opening during activated adenovirus E4 initiation and clearance. Using templates designed to restrict synthesis to defined positions, formation of a 3-nucleotide-long RNA was found to be sufficient to trigger expansion of the initial transcription bubble. This occurred by a discrete transition that expanded the downstream limit of melting from position 1 to 15. Subsequent clearance of the bubble from the promoter region also occurred without detectable intermediates. Thus, initial opening, extension, and the clearance of the promoter bubble appear to occur as discrete, unique transitions. The apparent K_m values for these three steps were determined to be near 5, 9, and 50 µM, respectively. Comparison of these values with ATPase activities within known transcription factors raises the possibility that different activities could be responsible for each step.

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