Detection of the protein-protein interaction between cyclic AMP receptor protein and RNA polymerase, by (13)C-carbonyl NMR.
Cyclic AMP receptor protein (CRP) plays a key role in the transcription regulation of many prokaryotic genes. Upon the binding of cyclic AMP, CRP is allosterically activated, binds to target DNA sites, and interacts with RNA polymerase. Although the protein-protein interaction between CRP and RNA polymerase is known to be important for the transcription initiation of the target genes, its structural understanding is still lacking, particularly due to the high molecular mass (approximately 120 kDa) of the protein complex. We assigned all of the (13)C-carbonyl resonances of methionine residues in CRP by using the double labeling and the enzyme digestion techniques. The result of (13)C-carbonyl NMR experiment on [(13)C'-Met]-CRP in the presence of both cyclic AMP and RNA polymerase alpha subunit showed that the two proteins 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 showed the effectiveness of the selective labeling and (13)C-carbonyl NMR spectroscopy in the specific detection of the protein-protein interaction between large molecules.
Lee
T W
TW
College of Pharmacy, Seoul National University, Seoul 151-742, Korea.
Won
H S
HS
Park
S H
SH
Kyogoku
Y
Y
Lee
B J
BJ
D000494:Allosteric Regulation; D001059:Apoproteins / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D002247:Carbon Isotopes; D002373:Cyclic AMP Receptor Protein / Q000737:chemistry* / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry* / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D008715:Methionine / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D008970:Molecular Weight; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021122:Protein Subunits
D001059:Apoproteins; D002247:Carbon Isotopes; D002373:Cyclic AMP Receptor Protein; D004268:DNA-Binding Proteins; D021122:Protein Subunits; D008715:Methionine; D012321:DNA-Directed RNA Polymerases; C113275:RNA polymerase alpha subunit