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Abstract: Bacteriophage T7 RNA polymerase undergoes major conformational changes as transcription proceeds from initiation to elongation. Using limited trypsin digestion and stopped-flow fluorescence kinetic methods, we have monitored promoter release, initial bubble collapse, and refolding of the 152–205 region (subdomain H), the latter being important for RNA channel formation. The kinetic studies show that the conformational changes are temporally coupled, commencing at the synthesis of 9 nt and completing by the synthesis of 12 nt of RNA. The temporal coupling of initial bubble collapse and RNA channel formation is proposed to facilitate proper binding of the RNA dissociated from the late initiation complexes into the RNA channel. Using promoter mutations, we have determined that promoter contacts are broken sequentially during transition from initiation to elongation. The specificity loop interactions are broken after synthesis of 8 nt or 9 nt of RNA, whereas the upstream promoter contacts persists up to synthesis of 12 nt of RNA. Both promoter contacts need to be broken for transition into elongation. The A-15C mutation resulted in efficient transition to elongation by synthesis of 9 nt of RNA, whereas the C-9A mutation resulted in early transition to elongation by synthesis of 7–8 nt of RNA. The effect of early promoter clearance in the mutant promoters was observed as reduced production of long abortive products. [Copyright &y& Elsevier]