Low energy CD of RNA hairpin unveils a loop conformation required for lambdaN antitermination activity.
N protein coded by phage lambda is a transcription factor that stimulates the antitermination activity of Escherichia coli RNA polymerase by binding specifically to the nascent RNA transcript at a stemloop structure called boxB. We use a new biophysical technique, involving the monitoring of the low energy circular dichroism spectra of 2-aminopurine residues site-specifically placed in the boxB RNA loop, to investigate this binding interaction. The low energy CD spectra of these 2-aminopurine probes reflect specific asymmetric interactions with adjacent nucleotide bases. Consequently, these CD spectra provide detailed and specific conformational information about the RNA chain at these chromophores that cannot be obtained from changes in the related fluorescence signals of these probes. CD changes were observed on binding the N peptide to boxB RNA that correspond to structural changes that had been previously seen by NMR, thus validating our experimental approach. The low energy CD method was then used to quantify the ordered and disordered states of the free hairpin loop and to show that a significant fraction of the boxB loop assumes a product-like structure in the absence of protein. A boxB derivative with an intact stem and a reduced concentration of ordered loop was identified and used to show that the extent of the reaction between protein and boxB depends on the concentration of structured loop in the RNA reactant population. This result has general implications for the conformational specificity of RNA-protein interactions.
Johnson
Neil P
NP
Institut de Pharmacologie et de Biologie Structurale, Toulouse, France. Neil.Johnson@ipbs.fr
Baase
Walter A
WA
von Hippel
Peter H
PH
D015075:2-Aminopurine / Q000737:chemistry; D001426:Bacterial Proteins / Q000737:chemistry*; D001703:Biophysics / Q000379:methods; D002942:Circular Dichroism / Q000379:methods*; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry; D004926:Escherichia coli / Q000201:enzymology / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D008856:Microscopy, Fluorescence; D008956:Models, Chemical; D009690:Nucleic Acid Conformation; D009841:Oligonucleotides / Q000737:chemistry; D010455:Peptides / Q000737:chemistry; D011487:Protein Conformation; D012313:RNA / Q000737:chemistry*; D012330:RNA, Double-Stranded / Q000737:chemistry; D016601:RNA-Binding Proteins / Q000737:chemistry*; D013696:Temperature
D001426:Bacterial Proteins; D009841:Oligonucleotides; D010455:Peptides; D012330:RNA, Double-Stranded; D016601:RNA-Binding Proteins; C064872:antiterminator proteins, Bacteria; D015075:2-Aminopurine; D012313:RNA; D012321:DNA-Directed RNA Polymerases