Identification of the RNA polymerase (RNAP) binding site for ppGpp, a central regulator of bacterial transcription, is crucial for understanding its mechanism of action. A recent high-resolution X-ray structure defined a ppGpp binding site on Thermus thermophilus RNAP. We report here effects of ppGpp on 10 mutant Escherichia coli RNAPs with substitutions for the analogous residues within 3-4 A of the ppGpp binding site in the T. thermophilus cocrystal. None of the substitutions in E. coli RNAP significantly weakened its responses to ppGpp. This result differs from the originally reported finding of a substitution in E. coli RNAP eliminating ppGpp function. The E. coli RNAPs used in that study likely lacked stoichiometric amounts of omega, an RNAP subunit required for responses of RNAP to ppGpp, in part explaining the discrepancy. Furthermore, we found that ppGpp did not inhibit transcription initiation by T. thermophilus RNAP in vitro or shorten the lifetimes of promoter complexes containing T. thermophilus RNAP, in contrast to the conclusion in the original report. Our results suggest that the ppGpp binding pocket identified in the cocrystal is not the one responsible for regulation of E. coli ribosomal RNA transcription initiation and highlight the importance of inclusion of omega in bacterial RNAP preparations.