VARIATION IN ATP REQUIREMENT DURING INFLUENZA-VIRUS TRANSCRIPTION

The ATP requirement of influenza A virus RNA-dependent RNA polymerase was studied during in vitro transcription reactions. In complete trans cription reactions, the K-m for ATP was 10-fold higher than the K-m va lues for the other NTPs. However, during transcription elongation the K-m for ATP was as low as the K-m values for the other NTPs, suggestin g a special requirement for ATP during transcription initiation. Gel a nalysis of RNA products of transcription initiation reactions showed t hat the incorporation of AMP into nascent RNA was more efficient at po sitions 4, 6 and 7 relative to the template RNA than at position 5. Th e polymerase produced short, abortive transcripts with lengths corresp onding to positions 3 and 4 relative to the template but never to posi tion 5 or longer. These results suggest that incorporation of AMP at p osition 5 induces the influenza A virus polymerase to go through a tra nsition from a transcription initiation to an elongation complex. This functional change of the polymerase complex rather than a requirement for ATP beta-gamma bond hydrolysis is the most likely reason for the particularly high K-m for ATP during the early phase of transcription. This conclusion is supported by the fact that the ATP analogue ATP ga mma S [adenosine 5'-O-(3-thiotriphosphate)] can efficiently replace AT P in in vitro transcription reactions and shows a comparable drop of K -m between transcription initiation and elongation.