Cooperative unfolding of Escherichia coli ribosome recycling factor originating from its domain–domain interaction and its implication for function
Section snippets
Bacterial strains, plasmids, and chemicals
Escherichia coli LJ14 is a MC1061 strain (frrts), of which the chromosomal wild-type frr allele is replaced with a mutant allele (V117D) [4], allowing E. coli LJ14 to grow at 30 °C but not at 42 °C. The strain was used for the in vivo activity assays of E. coli wild-type RRF (EcoRRF) and mutant RRFV117D by complementation analysis. E. coli BL21(DE3) plysS strain was used as a host cell for expression of EcoRRF and mutant RRFV117D. pET-DB was constructed in our laboratory [27] and used for the
Effect of V117D mutation on the CD spectra of E. coli RRF
Far-UV CD spectra were used to assess the secondary structure, especially the α-helical content. The appearance of two negative peaks at 208 and 222 nm in the CD spectrum is usually considered to be indicative of the content of α-helical structure in a protein [36], [37]. Fig. 2A shows the far-UV CD spectra of EcoRRF and mutant RRFV117D. The double negative peaks centered at 208 and 222 nm remained with slight decrease of the signal (8%) caused by V117D substitution, indicating that mutation has
Discussion
As described in the results section, although V117D substitution at domain I has no significant effect on the secondary structure of the RRF protein characterized by far-UV CD spectra (Fig. 2A), the mutation caused a decrease in the [θ]277nm value of 47% (Fig. 2B). This observation indicates that V117D mutation at domain I significantly perturbs the specific packing of tyrosine residues in the tertiary structure of mutant RRFV117D. Since all the tyrosine residues are located at domain II, the
Acknowledgments
We thank Professor A. Kaji, Department of Microbiology, School of Medicine, University of Pennsylvania, PA, USA for providing E. coli LJ14 and MRE600 strains. This work was supported by Grants (Nos. G1999075608 and 3017021) from the Chinese Committee for Science and Technology.
References (41)
- et al.
Cell
(2001) - et al.
Mol. Cell
(2002) - et al.
FEBS Lett.
(1999) - et al.
Adv. Biophys.
(1996) - et al.
Biochimie
(1996) - et al.
Mol. Cell
(1999) - et al.
J. Biol. Chem.
(2003) - et al.
J. Mol. Biol.
(2005) - et al.
Cell
(2002) - et al.
J. Biol Chem.
(2003)
Mol. Cell
Mol. Cell
Mol. Cell
Biochim. Biophys. Acta
J. Mol. Biol.
Arch. Biochem. Biophys.
Biophys. chem.
EMBO J.
EMBO J.
EMBO J.
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