Transcription activation at class II CRP-dependent promoters: Identification of determinants in the C-terminal domain of the RNA polymerase α subunit

Many transcription factors, including the Escherichia coli cyclic AMP receptor protein (CRP), act by making direct contacts with RNA polymerase. At Class II CRP-dependent promoters, CRP activates transcription by making two such contacts: (i) an interaction with the RNA polymerase α subunit C-terminal domain (αCTD) that facilitates initial binding of RNA polymerase to promoter DNA; and (ii) an interaction with the RNA polymerase α subunit N-terminal domain that facilitates subsequent promoter opening. We have used random mutagenesis and alanine scanning to identify determinants within αCTD for transcription activation at a Class II CRP-dependent promoter. Our results indicate that Class II CRP-dependent transcription requires the side chains of residues 265, 271, 285-288 and 317. Residues 285-288 and 317 comprise a discrete 20 x 10 Å surface on αCTD, and substitutions within this determinant reduce or eliminate cooperative interactions between α subunits and CRP, but do not affect DNA binding by α subunits. We propose that, in the ternary complex of RNA polymerase, CRP and a Class II CRP-dependent promoter, this determinant in αCTD interacts directly with CRP, and is distinct from and on the opposite face to the proposed determinant for αCTD-CRP interaction in Class I CRP-dependent transcription.