Requirement for the beta,gamma-pyrophosphate bond of ATP in a stage between transcription initiation and elongation by Escherichia coli RNA polymerase.

A linear fragment of DNA was fixed to acrylamide or agarose beads by its ends. When a fragment containing the lambda PR promoter is immobilized and transcribed, the RNA products are unchanged from those obtained on the unfixed DNA. Transcription from the immobilized fragment can be interrupted by diluting the reaction mixture into a large volume of the same buffer. Brief centrifugation allows isolation of the transcription complex with the immobilized DNA. If interruption occurs during elongation, the elongation can be resumed upon a second addition of substrates. If ATP is replaced by a beta, gamma-unhydrolyzable analogue in the second addition, the elongated products are similar to those obtained when the substrate contain ATP. When ATP is replaced by the analogue at the initiation step, however, the yield of elongated products is decreased to less than one-sixth and that of short abortive products is increased. Thus the ATP analogues are good substrates once elongation has been established in the presence of ATP, but not good enough to get past a stage just after initiation in the absence of ATP. We conclude that the beta, gamma-pyrophosphate bond of ATP is important for preparation of efficient elongation.