Significance Transcription initiation by RNA polymerase (RNAP) is a highly regulated rate-limiting step in many genes and involves numerous intermediate states that remain incompletely understood. Here, we report the characterization of a previously hypothesized slow initiation pathway involving RNAP backtracking and pausing. This backtracked and paused state is observed when all nucleoside triphosphates (NTPs) are present at physiologically relevant concentrations, but becomes more prevalent with unbalanced NTP levels, which may occur in vivo under conditions of metabolic stress. Pausing and backtracking in initiation may play an important role in regulating RNAP transcription. Moreover, similar RNA backtracked states may contribute to promoter-proximal pausing among eukaryotic RNA polymerase II enzymes.