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- Mehrdimensionale NMR-Spektroskopie (1) (remove)
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Structural insights into RNA binding by NusA and interaction studies of Nun with E. coli Nus factors.
(2008)
- In phage lambda, antitermination is initiated by the lambda-encoded N protein which recruits a number of host proteins called Nus factors. Several of these host proteins which are essential for effective transcription termination and antitermination have been identified, these includes NusA, NusB, NusE and NusG. The subject of this work is mainly focused on characterization of interactions between various Nus host factors in the termination and antitermination system by NMR spectroscopy. Like N protein, Nun also requires additional host factors for efficient termination. It has been reported already that NusA interacts with C-terminal region of Nun and also that Nun binding to NusA requires NusA ar1 region. On the basis of these results, 1H,15N-HSQC spectra were recorded to monitor the interaction between HK022-Nun and NusA ar1. NMR titration experiments between Nun and NusA clearly showed a lack of chemical shift perturbations. Therefore, it can be concluded that there is no intermolecular interaction between Nun and NusA ar1. Up to date, no information about the interaction between Nun and NusG as well as Nun and NusB are known. Titration experiments between Nun and both Nus factors, have also revealed no direct interaction. Altogether, it can be deduced that there might be no direct interaction between Nun and NusA ar1, and NusG, and NusB. The NusA transcription elongation protein, which binds nut site RNA, contains sequences corresponding to the S1 and KH classes of identified RNA binding domains. To gain comprehensive insights into binding surface on SKK domain upon nut RNA binding, backbone resonances of SKK domain was assigned using sequential C-alpha, C-beta and CO chemical shift information derived from an array of TROSY based triple-resonance experiments. With virtually complete backbone assignment (80.5 %) of the SKK domain it was possible to characterize the interaction between SKK domain and lambda nutL RNA by NMR titration experiments. Significant chemical shift changes observed on SKK domain upon addition of unlabeled lambda nutL RNA, had reflected a direct interaction. Mapping of chemical shift perturbations on SKK domain revealed that the RNA binding interface is mainly located in the KH domains. The results implied a sequence-specific RNA binding. In the free state, NusA cannot bind to RNA. Once alpha-CTD of RNA polymerase is bound to NusA the RNA binding inhibition is released. Direct interactions between alpha-CTD and NusA ar2 have been reported and therefore NusA ar2 could be a prime candidate for inhibiting the RNA binding of NusA. To further evaluate the autoinhibition effect of NusA ar2 on SKK domain, titrations between NusA ar2 and SKK domain have been performed. Upon NusA ar2 binding, notable chemical shift changes were observed in the KH1 region of SKK domain. The residues which were affected on binding to NusA ar2 were also affected during the SKK and lambda nutL titration experiments. The results are in good agreement with the proposed idea that NusA ar2 possibly occludes the RNA binding domains of NusA. To investigate the effect of alpha-CTD on RNA binding by NusA, titration of the complex containing SKK domain and NusA ar2 by gradually adding an increased molar ratio of alpha-CTD have been carried out. On addition of alpha-CTD, it was clearly observed that alpha-CTD displaced NusA ar2 from the complex suggesting that the inhibition of RNA binding by NusA ar2 could be released by alpha-CTD.
