ABSTRACT
Many transcription factors activate by directly interacting with RNA polymerase (RNAP). The C terminus of the RNAP alpha subunit (alphaCTD) is a common target of activators. We used both random mutagenesis and alanine scanning to identify alphaCTD residues that are crucial for MetR-dependent activation of metE and metH. We found that these residues localize to two distinct faces of the alphaCTD. The first is a complex surface consisting of residues important for alpha-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 alpha required for MetR-dependent activation. We have designed an in vitro reconstitution-purification protocol that allows us to specifically orient wild-type or mutant alpha subunits to either the beta-associated or the beta'-associated position within RNAP (comprising alpha(2), beta, beta', and sigma subunits). In vitro transcriptions using oriented alpha RNAP indicate that a single alphaCTD on either the beta- or the beta'-associated alpha subunit is sufficient for MetR activation of metE, while MetR interacts preferentially with the alphaCTD on the beta-associated alpha subunit at metH. We propose that the different alphaCTD 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 alphaCTD.
