Bacterial promoters are recognized by RNA polymerase (RNAP) σ subunit, which specifically interacts with the -10 and -35 promoter elements. Here, we provide evidence that the β' zipper, an evolutionarily conserved loop of the largest subunit of RNAP core, interacts with promoter spacer, a DNA segment that separates the -10 and -35 promoter elements, and facilitates the formation of stable closed promoter complex. Depending on the spacer sequence, the proposed interaction of the β' zipper with the spacer can also facilitate open promoter complex formation and even substitute for interactions of the σ subunit with the -35 element. These results suggest that there exists a novel class of promoters that rely on interaction of the β' zipper with promoter spacer, along with or instead of interactions of σ subunit with the -35 element, for their activity. Finally, our data suggest that sequence-dependent interactions of the β' zipper with DNA can contribute to promoter-proximal σ-dependent RNAP pausing, a recently recognized important step of transcription control.