Fine tuning of the E. coli NusB:NusE complex affinity to BoxA RNA is required for processive antitermination.
Phage lambda propagation in Escherichia coli host cells requires transcription antitermination on the lambda chromosome mediated by lambdaN protein and four host Nus factors, NusA, B, E (ribosomal S10) and G. Interaction of E. coli NusB:NusE heterodimer with the single stranded BoxA motif of lambdanutL or lambdanutR RNA is crucial for this reaction. Similarly, binding of NusB:NusE to a BoxA motif is essential to suppress transcription termination in the ribosomal RNA (rrn) operons. We used fluorescence anisotropy to measure the binding properties of NusB and of NusB:NusE heterodimer to BoxA-containing RNAs differing in length and sequence. Our results demonstrate that BoxA is necessary and sufficient for binding. We also studied the gain-of-function D118N NusB mutant that allows lambda growth in nusA1 or nusE71 mutants. In vivo lambda burst-size determinations, CD thermal unfolding measurements and X-ray crystallography of this as well as various other NusB D118 mutants showed the importance of size and polarity of amino acid 118 for RNA binding and other interactions. Our work suggests that the affinity of the NusB:NusE complex to BoxA RNA is precisely tuned to maximize control of transcription termination.
Lehrstuhl Biopolymere und Forschungszentrum für Bio-Makromoleküle, Universität Bayreuth, Bayreuth, Germany.
Björn M
BM
Burmann
Xiao
X
Luo
Paul
P
Rösch
Markus C
MC
Wahl
Max E
ME
Gottesman
D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D010582:Bacteriophage lambda / Q000235:genetics*; D001665:Binding Sites; D019281:Dimerization; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism*; D005454:Fluorescence Polarization; D009154:Mutation; D011485:Protein Binding; D012367:RNA, Viral / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D012269:Ribosomal Proteins / Q000737:chemistry / Q000378:metabolism*; D013489:Suppression, Genetic; D014157:Transcription Factors / Q000737:chemistry / Q000378:metabolism*
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; C074952:NusB protein, E coli; D012367:RNA, Viral; D016601:RNA-Binding Proteins; D012269:Ribosomal Proteins; D014157:Transcription Factors; C064872:antiterminator proteins, Bacteria; C029537:ribosomal protein S10