Promoter recognition by RNA polymerase is a key point in gene expression and a target of regulation. Bacterial RNA polymerase binds promoters in the form of the holoenzyme, with the σ specificity subunit being primarily responsible for promoter recognition. Free σ, however, does not recognize promoter DNA, and it has been proposed that the intrinsic DNA binding ability is masked in free σ but becomes unmasked in the holoenzyme. Here, we use a newly developed fluorescent assay to quantitatively study the interactions of free σ70 from Escherichia coli, the β′-σ complex, and the σ70 RNA polymerase (RNAP) holoenzyme with non-template strand of the open promoter complex transcription bubble in the context of model non-template oligonucleotides and fork junction templates. We show that σ70, free or in the context of the holoenzyme, recognizes the -10 promoter element with the same efficiency and specificity. The result implies that there is no need to invoke a conformational change in σ for recognition of the -10 element in the single-stranded form. In the holoenzyme, weak but specific interactions of σ are increased by contacts with DNA downstream of the -10 element. We further show that region 1 of σ70 is required for stronger interaction with non-template oligonucleotides in the holoenzyme but not in free σ. Finally, we show that binding of the β′ RNAP subunit is sufficient to allow specific recognition of the TG motif of the extended -10 promoter element by σ70. The new fluorescent assay, which we call a protein beacon assay, will be instrumental in quantitative dissection of fine details of RNAP interactions with promoters.