Transcription initiation is a dynamic process in which RNA polymerase (RNAP) and promoter DNA act as partners, changing in response to one another, to produce a polymerase/promoter open complex (RPo) competent for transcription. In Escherichia coli RNAP, region 1.1, the N-terminal 100 residues of σ⁷⁰, is thought to occupy the channel that will hold the DNA downstream of the transcription start site; thus, region 1.1 must move from this channel as RPo is formed. Previous work has also shown that region 1.1 can modulate RPo formation depending on the promoter. For some promoters region 1.1 stimulates the formation of open complexes; at the P_minor promoter, region 1.1 inhibits this formation. We demonstrate here that the AT-rich P_minor spacer sequence, rather than promoter recognition elements or downstream DNA, determines the effect of region 1.1 on promoter activity. Using a P_minor derivative that contains good σ⁷⁰-dependent DNA elements, we find that the presence of a more GC-rich spacer or a spacer with the complement of the P_minor sequence results in a promoter that is no longer inhibited by region 1.1. Furthermore, the presence of the P_minor spacer, the GC-rich spacer, or the complement spacer results in different mobilities of promoter DNA during gel electrophoresis, suggesting that the spacer regions impart differing conformations or curvatures to the DNA. We speculate that the spacer can influence the trajectory or flexibility of DNA as it enters the RNAP channel and that region 1.1 acts as a “gatekeeper” to monitor channel entry.