Interaction of the intrinsically unstructured phage lambda N Protein with Escherichia coli NusA.
N protein of the Escherichia coli phage lambda (lambdaN) is involved in antitermination, a transcription regulatory process that is essential for the expression of delayed early genes during phage lytic development. lambdaN is an intrinsically unstructured protein that possesses three distinct binding sites interacting with the carboxy terminus of the E. coli host factor protein NusA, the viral nutBoxB-RNA, and RNA polymerase, respectively. Heteronuclear NMR experiments with lambdaN(1-53) in complex with NusA(339-495) revealed that upon complex formation the lambdaN-binding interface, lambdaN(34-47), adopts a rigid structure. NMR data also indicate the induction of a weak helical structure in the nutboxB RNA-binding region lambdaN(1-22) upon binding to NusA(339-495) even in the absence of RNA. Titration experiments of the lambdaN(1-53)-nutBoxB RNA complex with NusA(339-495) revealed that the ternary complex can be described in terms of two structurally independent binary interactions. Furthermore, chemical-shift perturbation experiments with different NusA constructs and different lambdaN peptides showed that only NusA(353-416) is involved in lambdaN binding. We found that only one molecule of NusA(339-426) binds to one molecule of lambdaN(1-53). We also clarified the role of the lambdaN-binding region and could show that N41-R47 also binds to NusA(339-495). Furthermore, we observe that lambdaN(1-22) adopts a helical fold upon binding to NusA(339-495), in agreement with one of the theoretical models of lambdaN action.
Lehrstuhl Biopolymere, Universität Bayreuth, Universitaetsstrasse 30, 95440 Bayreuth, Germany.
Stefan
S
Prasch
Sabine
S
Schwarz
Anke
A
Eisenmann
Birgitta M
BM
Wöhrl
Kristian
K
Schweimer
Paul
P
Rösch
D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000378:metabolism*; D019906:Nuclear Magnetic Resonance, Biomolecular; D010445:Peptide Elongation Factors / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism; D011485:Protein Binding; D017433:Protein Structure, Secondary; D014157:Transcription Factors / Q000378:metabolism*; D035602:Transcriptional Elongation Factors; D054334:Viral Regulatory and Accessory Proteins / Q000378:metabolism*
D029968:Escherichia coli Proteins; C059417:N protein, Bacteriophage lambda; D010445:Peptide Elongation Factors; D010446:Peptide Fragments; D014157:Transcription Factors; D035602:Transcriptional Elongation Factors; D054334:Viral Regulatory and Accessory Proteins; C068727:nusA protein, E coli