Nucleic Acids Research Advance Access published online on October 23, 2009
Nucleic Acids Research, doi:10.1093/nar/gkp736
Structural Biology |
Fine tuning of the E. coli NusB:NusE complex affinity to BoxA RNA is required for processive antitermination
1Lehrstuhl Biopolymere und Forschungszentrum für Bio-Makromoleküle, Universität Bayreuth, Universitätsstraße 30, 95447 Bayreuth, 2Max-Planck-Institut für biophysikalische Chemie, Makromolekulare Röntgenkristallographie, Am Faßberg 11, 37077 Göttingen, 3Freie Universität Berlin, Fachbereich Biologie-Chemie-Pharmazie, Institut für Chemie und Biochemie, AG Strukturbiochemie, Takustr. 6, 14195 Berlin, Germany and 4Department of Microbiology and Institute of Cancer Research, Columbia University Medical Center, New York, NY 10032, USA
*To whom correspondence should be addressed. Tel: +49 921 55 3540; Fax: +49 921 55 3544; Email: roesch{at}unibt.de
Received July 22, 2009. Revised August 20, 2009. Accepted August 20, 2009.
Phage 
propagation in Escherichia coli host cells requires transcription antitermination on the chromosome mediated by N 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 nutL or nutR 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 growth in nusA1 or nusE71 mutants. In vivo 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.