Many transcription factors activate by directly interacting with RNA polymerase (RNAP). The C terminus of the RNAP  subunit (CTD) is a common target of activators. We used both random mutagenesis and alanine scanning to identify CTD residues that are crucial for MetR-dependent activation of metE and metH. We found that these residues localize to two distinct faces of the CTD. The first is a complex surface consisting of residues important for -DNA interactions, activation of both genes (residues 263, 293, and 320), and activation of either metE only (residues 260, 276, 302, 306, 309, and 322) or metH only (residues 258, 264, 290, 294, and 295). The second is a distinct cluster of residues important for metE activation only (residues 285, 289, 313, and 314). We propose that a difference in the location of the MetR binding site for activation at these two promoters accounts for the differences in the residues of  required for MetR-dependent activation. We have designed an in vitro reconstitution-purification protocol that allows us to specifically orient wild-type or mutant  subunits to either the -associated or the '-associated position within RNAP (comprising ₂, , ', and  subunits). In vitro transcriptions using oriented  RNAP indicate that a single CTD on either the - or the '-associated  subunit is sufficient for MetR activation of metE, while MetR interacts preferentially with the CTD on the -associated  subunit at metH. We propose that the different CTD requirements at these two promoters are due to a combination of the difference in the location of the activation site and limits on the rotational flexibility of the CTD.