Spectroscopic and thermodynamic characterization of the transcription antitermination factor NusE and its interaction with NusB from Mycobacterium tuberculosis

N-utilizing proteins (Nus) form a complex involved in the regulation of rRNA biosynthesis in enteric bacteria by modulating the efficiency of transcriptional termination [Nodwell, J. R., and Greenblatt, J. (1993) Cell 72, 261-268]. The protein NusE (identical to the protein S10 of the small ribosomal subunit) from the pathogenic mycobacterium M. tuberculosis has been cloned and overexpressed in Escherichia coli. The pure protein has been characterized by circular dichroism, ultracentrifugation, NMR, and binding to NusB. The near-ultraviolet circular dichroism spectrum of this protein suggests that it has a moderate (ca. 12-16%) alpha -helical content at 30 degreesC. The protein undergoes cold denaturation, with a temperature of maximum stability near 30 degreesC, implying a substantial heat capacity difference between the folded and unfolded states. The sedimentation equilibrium and velocity data indicate that the protein is monomeric and expanded in solution. NMR spectroscopy shows that there is no significant tertiary structure, and confirms the low secondary structure content at low temperatures. Furthermore, then was evidence for more structure at 30 degreesC than at 10 degreesC. Well-defined shifts in peaks in the HSQC spectrum of N-15 labeled NusE/NusB when the unlabeled counterpart was added at approximately stoichiometric concentrations showed the formation of a NusE-NusB complex in the absence of RNA. The far-UV CD and ultracentrifuge experiments, however, indicated relatively weak binding. Isothermal titration calorimetry showed the binding was weak and endothermic at 15 degreesC, with a total DeltaH of greater than or equal to 10 kcal/mol, This weak binding is consistent with a small interaction interface and lack of large conformational rearrangements in the predominantly unfolded NusE protein. The conformational flexibility of NusE may be important for its roles in both the ribosome and antitermination complexes.