Spectroscopic and Thermodynamic Characterization of the Transcription Antitermination Factor NusE and Its Interaction with NusB from Mycobacterium tuberculosis
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- 作者：B. Gopal ; K. G. Papavinasasundaram ; Guy Dodson ; M. Jo Colston ; Sarah A. Major ; and Andrew N. Lane
- 刊名：Biochemistry
- 出版年：2001
- 出版时间：January 30, 2001
- 年：2001
- 卷：40
- 期：4
- 页码：920 - 928
- 全文大小：1010K
- 年卷期：v.40,no.4(January 30, 2001)
- ISSN：1520-4995
文摘
N-utilizing proteins (Nus) form a complex involved in the regulation of rRNA biosynthesis inenteric 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 Escherichiacoli. The pure protein has been characterized by circular dichroism, ultracentrifugation, NMR, and bindingto NusB. The near-ultraviolet circular dichroism spectrum of this protein suggests that it has a moderate(ca. 12-16%) 
ges/gifchars/alpha.gif" BORDER=0>-helical content at 30 ges/entities/deg.gif">C. The protein undergoes cold denaturation, with a temperature ofmaximum stability near 40 ges/entities/deg.gif">C, implying a substantial heat capacity difference between the folded andunfolded states. The sedimentation equilibrium and velocity data indicate that the protein is monomericand expanded in solution. NMR spectroscopy shows that there is no significant tertiary structure, andconfirms the low secondary structure content at low temperatures. Furthermore, there was evidence formore structure at 30 ges/entities/deg.gif">C than at 10 ges/entities/deg.gif">C. Well-defined shifts in peaks in the HSQC spectrum of 15N labeledNusE/NusB when the unlabeled counterpart was added at approximately stoichiometric concentrationsshowed the formation of a NusE-NusB complex in the absence of RNA. The far-UV CD andultracentrifuge experiments, however, indicated relatively weak binding. Isothermal titration calorimetryshowed the binding was weak and endothermic at 15 ges/entities/deg.gif">C, with a total ges/gifchars/Delta.gif" BORDER=0 >H of ges/entities/ge.gif">10 kcal/mol. This weakbinding is consistent with a small interaction interface and lack of large conformational rearrangementsin the predominantly unfolded NusE protein. The conformational flexibility of NusE may be importantfor its roles in both the ribosome and antitermination complexes.