RNA-protein recognition: single-residue ultrafast dynamical control of structural specificity and function.
The transcription antiterminator N protein from bacteriophage lambda uses its arginine-rich motif to specifically bind a stem-loop RNA hairpin (boxB) as a bent alpha-helix. A single stacking interaction between a tryptophan (Trp-18) and an adenosine (A7) in the RNA loop is robust and necessary for antitermination activity in vivo. Previously, femtosecond fluorescence up-conversion experiments from this laboratory indicated that the N/boxB complex exists in a dynamical two-state equilibrium between stacked and unstacked conformations and that the extent of stacking depends on the identity of peptide residues 14 and 15. In the present work, we have combined transient absorption and fluorescence up-conversion to determine the nature of interactions responsible for this sequence-dependent behavior. Analysis of mutant complexes supports the idea that the beta-carbon of residue 14 enforces the stacked geometry by hydrophobic interaction with the ribose of A7, whereas a positive charge at this residue plays only a secondary role. A positive charge at position 15 substantially disfavors the stacked state but retains much of the binding energy. Remarkably, in vivo antitermination experiments show strong correlation with our femtosecond dynamics, demonstrating how conformational interplay can control the activity of a macromolecular machine.
Xia
Tianbing
T
Laboratory for Molecular Sciences, Arthur Amos Noyes Laboratory of Chemical Physics, California Institute of Technology, Pasadena, CA 91125, USA.
Wan
Chaozhi
C
Roberts
Richard W
RW
Zewail
Ahmed H
AH
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D057927:Hydrophobic and Hydrophilic Interactions; D007834:Lasers; D011487:Protein Conformation; D012313:RNA / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D013057:Spectrum Analysis; D013997:Time Factors; D014158:Transcription, Genetic; D054334:Viral Regulatory and Accessory Proteins / Q000737:chemistry / Q000378:metabolism*
C059417:N protein, Bacteriophage lambda; D016601:RNA-Binding Proteins; D054334:Viral Regulatory and Accessory Proteins; D012313:RNA