Cooperative unfolding of Escherichia coli ribosome recycling factor originating from its domain–domain interaction and its implication for function

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Abstract

Cooperative unfolding of Escherichia coli ribosome recycling factor (RRF) and its implication for function were investigated by comparing the in vitro unfolding and the in vivo activity of wild-type E. coli RRF and its temperature-sensitive mutant RRFV117D. The experiments show that mutation V117D at domain I could perturb the domain II structure as evidenced in the near-UV CD and tyrosine fluorescence spectra though no significant globular conformation change occurred. Both equilibrium unfolding induced by heat or denaturant and kinetic unfolding induced by denaturant obey the two-state transition model, indicating V117D mutation does not perturb the efficient interdomain interaction, which results in cooperative unfolding of the RRF protein. However, the mutation significantly destabilizes the E. coli RRF protein, moving the thermal unfolding transition temperature range from 50–65 to 35–50 °C, which spans the non-permissive temperature for the growth of E. coli LJ14 strain (frrts). The in vivo activity assays showed that although V117D mutation results in a temperature sensitive phenotype of E. coli LJ14 strain (frrts), over-expression of mutant RRFV117D can eliminate the temperature sensitive phenotype at the non-permissive temperature (42 °C). Taking all the results into consideration, it can be suggested that the mechanism of the temperature sensitive phenotype of the E. coli LJ14 cells is due to inactivation of mutant RRFV117D caused by unfolding at the non-permissive temperatures.

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.

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