TRANSCRIPTION ACTIVATION AT CLASS-II CRP-DEPENDENT PROMOTERS - IDENTIFICATION OF DETERMINANTS IN THE C-TERMINAL DOMAIN OF THE RNA-POLYMERASE ALPHA-SUBUNIT

Many transcription factors, including the Escherichia coli cyclic AMP receptor protein (CRP), act by making direct contacts with RNA polymer ase. At Class II CRP-dependent promoters, CRP activates transcription by making two such contacts: (i) an interaction with the RNA polymeras e alpha subunit C-terminal domain (alpha CTD) that facilitates initial binding of RNA polymerase to promoter DNA; and (ii) an interaction wi th the RNA polymerase alpha subunit N-terminal domain that facilitates subsequent promoter opening. We have used random mutagenesis and alan ine scanning to identify determinants within alpha CTD for transcripti on activation at a Class II CRP-dependent promoter, Our results indica te that Class II: CRP-dependent transcription requires the side chains of residues 265, 271, 285-288 and 317. Residues 285-288 and 317 compr ise a discrete 20 x 10 Angstrom surface on aCTD, and substitutions wit hin this determinant reduce or eliminate cooperative interactions betw een alpha subunits and CRP, but do not affect DNA binding by alpha sub units, We propose that, in the ternary complex of RNA polymerase, CRP and a Class II CRP-dependent promoter, this determinant in alpha CTD i nteracts directly with CRP, and is distinct from and on the opposite f ace to the proposed determinant for alpha CTD-CRP interaction in Class I CRP-dependent transcription.