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 transcription reactions, the K-m for ATP was 10-fold higher than the K-m values 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, suggesting a special requirement for ATP during transcription initiation. Gel analysis of RNA products of transcription initiation reactions showed that the incorporation of AMP into nascent RNA was more efficient at positions 4, 6 and 7 relative to the template RNA than at position 5. The polymerase produced short, abortive transcripts with lengths corresponding to positions 3 and 4 relative to the template but never to position 5 or longer. These results suggest that incorporation of AMP at position 5 induces the influenza A virus polymerase to go through a transition 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 gamma S [adenosine 5'-O-(3-thiotriphosphate)] can efficiently replace ATP in in vitro transcription reactions and shows a comparable drop of K-m between transcription initiation and elongation.