RNA Polymerase-cNMP-ligated cAMP Receptor Protein (CRP) Mutant Interactions in the Enhancement of Transcription by CRP Mutants

Abstract

The enhancement of the transcription of three synthetic promoters by cNMP-ligated cAMP receptor protein (CRP)/mutant complexes was determined from the transcription yields of a short AAUU transcript in an abortive initiation in vitro transcription assay. The cNMP-ligated CRP and mutants were cAMP, cGMP, and cIMP ligated with CRP, T127L CRP, S128A CRP, and T127L/S128A CRP. The transcriptional activation of a 152-base pair lacUV5 promoter (synlac promoter) with a CRP consensus binding site sequence (syncon promoter) was enhanced by an average factor of 12. The transcription of over 25 operons, which code for enzymes involved in carbohydrate metabolism, is enhanced by the binding of cAMP receptor protein (CRP) 1 to the promoter region of the operon at a site adjacent to the RNA polymerase binding site. Low glucose levels in the cell increase the level of cAMP, resulting in substantial cAMP binding to the amino-terminal domains of the CRP dimer (45,000 g mol Ϫ1 ), which induces a conformational change in the CRP so that the two carboxylterminal domains bind specifically to a site in the promoter (1), centered either 70.5, 61.5, or 41.5 base pairs upstream from the promoter transcription start point (P1). Mutations along the helical monomer-monomer interface of CRP significantly alter the in vivo level of transcriptional enhancement by CRP. Conversion of the helical interface to a more perfect leucine zipper by mutating Thr 127 to Leu (T127L) results in in vivo activation of transcription in the presence of cGMP, an analog of cAMP (2). Mutation of the Ser 128 residue, which interacts with cAMP in the other subunit, to Ala (S128A) reduces the enhancement of in vivo transcription by CRP (2, 3). A mutant of CRP with both mutations (CRP*) enhances in vivo transcription in the absence of cAMP (2). In addition, the mutation of Thr 127 to Cys, Ile, or Ser also resulted in the enhancement of in vivo and in vitro transcription in the presence of cGMP, and it was concluded that the Thr 127 3 Cys, Ile, and Ser mutations in CRP produced structural changes in CRP similar to those induced by cAMP binding to CRP (3). Since the binding affinities of cAMP to CRP and the CRP mutants are nearly the same (3, 4), changes in the enhancement of transcription by CRP must involve processes subsequent to the binding of cAMP to CRP. A recent isothermal titration calorimetric (ITC) study of the binding of three 40-bp DNA duplexes with each one containing the CRP binding site sequence of a promoter to cNMP-ligated CRP, T127L, S128A, and CRP* revealed large differences in the CRP binding site affinities, which could account for differences in the enhancement of transcription by the cNMP-ligated CRP mutants (5). In addition, fluorescence polarization studies show that CRP with bound DNA (6) also interacts with RNA polymerase. Photocross-linking studies indicate that CRP is in close enough proximity to interact with RNA polymerase on the lac promoter (7). Transcription results from surface mutations on CRP show that the most likely RNA polymerase...

Structural Basis of Transcription Activation: The CAP-αCTD-DNA Complex

“…The structure also provides a rationalization for genetic results indicating involvement of residues 162 and 163 of CAP and residues 289 and 290 of ␣CTD (1, 8 -11, 19); in the structure, these residues underlie (and make hydrogen bonds and van der Waals interactions with) residues of AR1 and the 287 determinant of ␣CTD and, as such, are likely to be critical determinants of the conformations of AR1 and the 287 determinant. The observed small size of the interface between CAP and ␣CTD CAP,DNA is in agreement with genetic results ruling out involvement of other residues (10,18,19) and with biochemical results indicating a modest magnitude of transcriptional activation (a factor of about 10 at P lac ) (34) and a modest magnitude of CAP-RNAP interaction free energy (about -1 to -2 kcal/mol at P lac ) (35)(36)(37).…”

Entropic Nature of the Interaction between Promoter Bound CRP Mutants and RNA Polymerase

“…ITC Measurements. The binding affinity, enthalpy, and entropy of the cNMP-ligated CRP/mutants to the 104 bp syncon promoter and the promoter−RNA polymerase complex were determined from ITC measurements, using a Microcal, Inc. VP Titration Calorimeter as described previously ( , ). The sample vessel contained either the RNA polymerase, the RNA polymerase−104 bp syncon promoter complex, or the promoter alone in the phosphate buffer, while the reference vessel contained just the buffer solution.…”

The Structure of the T127L/S128A Mutant of cAMP Receptor Protein Facilitates Promoter Site Binding

“…The T127L (7) and T127C (8) CRP mutants remove the specificity of the activation of CRP by cAMP so that CRP is also activated by the binding of cGMP. A CRP double mutant containing the T127L mutation and a S128A mutation (CRP*) activates in vivo and in vitro transcription in the absence of a cyclic nucleotide monophosphate (7,9). The Thr 127 mutations appear to alter the conformation of CRP to a conformation close to that of the allosterically activated CRP so that binding of the cAMP is not necessary (10).…”