Transcription activation at Class II CRP-dependent promoters: identification of determinants in the C-terminal domain of the RNA polymerase alpha subunit

Abstract

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 (alphaCTD) 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 alphaCTD 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 20x10 A surface on alphaCTD, 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 alphaCTD interacts directly with CRP, and is distinct from and on the opposite face to the proposed determinant for alphaCTD-CRP interaction in Class I CRP-dependent transcription.

Transcription Activation by CooA, the CO-sensing Factor fromRhodospirillum rubrum

“…In class II CRP-dependent promoters, ␣-CTD makes nonspecific contacts with the DNA segment immediately upstream of the CRP site (16,23). In this study, we found that wild-type RNAP, but not RNAP with the ␣-CTD truncation, extends DNase I protection upstream of that protected by CooA alone.…”

“…This also suggests that the upstream A ϩ T-rich sequence of P cooF does not act as a UP element. Although we cannot exclude the possibility that some portion of the protection upstream of the CooA-binding site is due to a conformational change in CooA induced by the presence of RNAP, the interaction of ␣-CTD with DNA upstream of CooA is consistent with the sequence-nonspecific interactions between DNA and ␣-CTD observed at class II CRP-dependent promoters (23).…”

“…These percentages represent the averages of three independent experiments in which all values were within 18% of the average. least one residue in ␣-CTD (Val 287 ) that has no effect on DNA binding (23) suggests that the ␣-CTD requirement involves, at least in part, a protein-protein interaction between CooA and ␣-CTD.…”

“…This interaction increases initial binding of RNAP to the promoter (22). Recently, residues 285-288 and 317 of ␣-CTD have been shown to comprise the surface that interacts with AR1 of CRP at class II promoters (23). The second contact, between activating region 2 (AR2) of the downstream subunit of CRP and the N-terminal domain of the ␣ subunit, facilitates isomerization of the closed complex to the open complex (24).…”

Exploitation of a Chemical Nuclease to Investigate the Location and Orientation of the Escherichia coli RNA Polymerase α Subunit C-terminal Domains at Simple Promoters That Are Activated by Cyclic AMP Receptor Protein

“…Analysis of the high resolution crystal structure of a CRP⅐␣CTD⅐DNA ternary complex shows that AR1 contacts a segment of the surface of ␣CTD that is located on the opposite face from the 261 determinant (8). This surface, known as the 287 determinant, includes residues 285-289 that had been identified in previous genetic analyses as being the most likely residues in ␣CTD to make direct contact with CRP (19,20).…”