Using limited proteolysis, we show that the Escherichia coli RNA polymerase alpha subunit consists of an N-terminal domain comprised of amino acids 8-241, a C-terminal domain comprised of amino acids 249-329, and an unstructured and/or flexible interdomain linker. We have carried out a detailed structural and functional analysis of an 85 amino acid proteolytic fragment corresponding to the C-terminal domain (alpha CTD-2). Our results establish that alpha CTD-2 has a defined secondary structure (approximately 40% alpha helix, approximately 0% beta sheet). Our results further establish that alpha CTD-2 is a dimer and that alpha CTD-2 exhibits sequence-specific DNA binding activity. Our results suggest a model for the mechanism of involvement of alpha in transcription activation by promoter upstream elements and upstream-binding activator proteins.