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 alpha subunit C-terminal domain (alpha CTD) that facilitates initial binding of RNA polymerase to promoter DNA; and (ii) an interaction with the RNA polymerase alpha subunit N-terminal domain that facilitates subsequent promoter opening. We have used random mutagenesis and alanine scanning to identify determinants within alpha CTD 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 Angstrom surface on aCTD, and substitutions within this determinant reduce or eliminate cooperative interactions between alpha subunits and CRP, but do not affect DNA binding by alpha subunits, We propose that, in the ternary complex of RNA polymerase, CRP and a Class II CRP-dependent promoter, this determinant in alpha CTD interacts directly with CRP, and is distinct from and on the opposite face to the proposed determinant for alpha CTD-CRP interaction in Class I CRP-dependent transcription.