Detection of the protein-protein interaction between cyclic AMP receptor protein and RNA polymerase, by C-13-carbonyl NMR

Cyclic AMP receptor protein (CRP) plays a key role in the transcription reg ulation of many prokaryotic genes. Upon the binding of cyclic AMP, CRP is a llosterically activated, binds to target DNA sites, and interacts with RNA polymerase, Although the protein-protein interaction between CRP and RNA po lymerase is known to be important for the transcription initiation of the t arget genes, its structural understanding is still lacking, particularly du e to the high molecular mass (similar to 120 M)a) of the protein complex. W e assigned all of the C-13-carbonyl resonances of methionine residues in CR P by using the double labeling and the enzyme digestion techniques. The res ult of C-13-carbonyl NMR experiment on [C-13'-Met]-CRP in the presence of b oth cyclic AMP and RNA polymerase alpha subunit showed that the two protein s interact with each other in solution in the absence of DNA via the region around the residues from Met 157 to Met 163 in CRP, The results also showe d the effectiveness of the selective labeling and C-13-carbonyl NMR spectro scopy in the specific detection of the protein-protein interaction between large molecules.