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Transcription activation at Class II CRP‐dependent promoters: identification of determinants in the C‐terminal domain of the RNA polymerase α subunit
Author(s) -
Savery Nigel J.,
Lloyd Georgina S.,
Kainz Mark,
Gaal Tamas,
Ross Wilma,
Ebright Richard H.,
Gourse Richard L.,
Busby Stephen J.W.
Publication year - 1998
Publication title -
the embo journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 7.484
H-Index - 392
eISSN - 1460-2075
pISSN - 0261-4189
DOI - 10.1093/emboj/17.12.3439
Subject(s) - biology , rna polymerase , transcription factor ii d , rna polymerase ii , transcription (linguistics) , promoter , microbiology and biotechnology , rna polymerase ii holoenzyme , general transcription factor , polymerase , transcription factor ii e , transcription factor ii f , transcription preinitiation complex , rna , genetics , dna , gene , gene expression , linguistics , philosophy
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 20×10 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.