During transcription initiation, RNA polymerases appear to retain promoter interactions while transcribing short RNAs that are frequently released from the complex. Upon transition to elongation, the polymerase releases promoter and forms a stable elongation complex. Little is known about the changes in polymerase conformation or polymerase:DNA interactions that occur during this process. To characterize the transitions that occur in the T7 RNA polymerase transcription complex during initiation, we prepared enzymes with Fe-BABE conjugated at 11 different positions. Addition of H(2)O(2) to transcription complexes prepared with these enzymes led to nucleic acid strand scission near the conjugate. Changes in the cleavage sites revealed a series of conformational changes and rearrangements of protein:nucleic acid contacts that mediate progression through the initiation reaction.