The DNA-dependent RNA polymerase (RNAP) from Escherichia coli has previously been reported to specifically initiate transcription from viroid-derived RNA promoters in vitro. In order to gain insight into the molecular mechanism of RNA promoter recognition by this RNAP, we have used nucleic acid intercalators and RNA:protein footprinting experiments to study the interaction between the polymerase and an RNA promoter at the initiation site. Our data revealed that the polymerase binds an external single-stranded loop, rather than a double-stranded region as is the case for DNA templates. Despite this divergence in promoter binding, the model RNA template was bound by both the beta and beta' subunits of the RNAP, as is observed with DNA templates. Most importantly, this work proposes large single-stranded RNA hairpin loops have the potential to be promoters for DNA-dependent RNAP. (C) 2004 Elsevier Inc. All rights reserved.