Amino acid residues in region 2 of {70} have been shown to play an important role in the strand separation step which is necessary for formation of the functional or open RNA polymerase- promoter complex. Here we present a comparison of the roles of basic and aromatic amino acids in the accomplishment of this process, using RNA polymerase bearing alanine substitutions for both types of amino acids in region 2. We determined the effects of the substitutions on the kinetics of open complex formation, as well as on the ability of the RNA polymerase to form complexes with single stranded DNA, and with forked DNA duplexes carrying a single stranded overhang consisting of bases in the -10 region. We conclude that two basic amino acids (K414 and K418) are important for promoter binding and demonstrate distinct roles, at a subsequent step, for two aromatic amino acids (Y430 and W433). It is likely that these 4 amino acids, which are close to each other in the structure of {70}, together are involved in the nucleation of the strand separation process.