Domain II plays a crucial role in the function of ribosome recycling factor

Abstract

RRF (ribosome recycling factor) consists of two domains, and in concert with EF-G (elongation factor-G), triggers dissociation of the post-termination ribosomal complex. However, the function of the individual domains of RRF remains unclear. To clarify this, two RRF chimaeras, EcoDI/TteDII and TteDI/EcoDII, were created by domain swaps between the proteins from Escherichia coli and Thermoanaerobacter tengcongensis. The ribosome recycling activity of the RRF chimaeras was compared with their wild-type RRFs by using in vivo and in vitro activity assays. Like wild-type TteRRF (T. tengcongensis RRF), the EcoDI/TteDII chimaera is non-functional in E. coli, but both wild-type TteRRF, and EcoDI/TteDII can be activated by coexpression of T. tengcongensis EF-G in E. coli. By contrast, like wild-type E. coli RRF (EcoRRF), TteDI/EcoDII is fully functional in E. coli. These findings suggest that domain II of RRF plays a crucial role in the concerted action of RRF and EF-G for the post-termination complex disassembly, and the specific interaction between RRF and EF-G on ribosomes mainly depends on the interaction between domain II of RRF and EF-G. This study provides direct genetic and biochemical evidence for the function of the individual domains of RRF.

Figure 1. (A) Comparison of the primary structures of EcoRRF with TteRRF

Amino acid sequences are shown as single letter codes. The numbering at the bottom is the amino acid sequence from T. thermophilus RRF. Identical residues are shadowed in black and similar residues are boxed. The corresponding secondary-structure elements of EcoRRF are indicated at the top. Vertical arrows indicate the points where the two domains were exchanged (G30 and R31, P103 and P104 for EcoRRF; G29 and R30, P102 and E103 for TteRRF respectively). (B) Schematic drawings of the structures of EcoRRF, TteRRF, TteDI/EcoDII, and EcoDI/TteDII.

Figure 2. Complementation analysis of E. coli LJ14 with RRFs

The E. coli LJ14 (frr^ts) strain was transformed with the respective plasmids and examined for growth-phenotype. (A) Growth curves of various transformants of E. coli LJ14 (frr^ts) at the permissive temperature (30 °C). Vector, transformants with pQE-60; EcoRRF, transformants with pQE-EcoRRF; EcoDI/TteDII, transformants with pQE-EcoDI/TteDII; TteDI/DII, transformants with pQE-TteDI/EcoDII; TteRRF, transformants with pQE-TteRRF. (B) Growth curves of various transformants of E. coli LJ14 (frr^ts) at the non-permissive temperature (42 °C). The transformants are the same as those indicated in (A). (C) Detection of RRF expression in E. coli LJ14 by immunoblotting using anti-EcoRRF antibodies. Protein extracts were prepared from transformants harbouring the respective plasmids. Bands: 1, pQE-60 vector; 2, PQE-EcoRRF; 3, pQE-TteRRF; 4, pQE-EcoDI/TteDII; 5, pQE-TteDI/EcoDII. (D) Growth curves of various transformants of E. coli LJ14 (frr^ts) at the non-permissive temperature (42 °C). Vectors, transformants with pQE-60 and pSTV-28; TteEF-G, transformants with pQE-60 and pSTV-TteEF-G; TteRRF, transformants with pQE-TteRRF and pSTV-28; TteRRF and Tte-EFG, transformants with pQE-TteRRF and pSTV-TteEF-G. (E) Growth curves of various transformants of E. coli LJ14 (frr^ts) at the non-permissive temperature (42 °C). Vectors, transformants with pQE-60 and pSTV-28; Tte-EFG, transformants with pQE-60 and pSTV-TteEF-G; EcoDI/TteDII, transformants with pQE-EcoDI/TteDII and pSTV-28; EcoDI/TteDII&Tte-EFG, transformants with pQE-EcoDI/TteDII and pSTV-TteEF-G. The experimental conditions are described in the text.

Figure 3. Polysome-breakdown assays of RRFs

Reactions were set up as described in the Materials and methods section. (A) Polysomes alone; (B) left column, RRFs with EcoEF-G; right column, RRFs with TteEF-G. Each reaction mixture contains 30 μg of RRF and 60 μg of EcoEF-G or TteEF-G.

Figure 4. EF-Gs release RRFs from 70 S ribosomes in a dose-dependent manner

Complexes of EcoRRF, EcoDI/TteDII, TteDI/EcoDII or TteRRF (4 μM) with 70 S ribosomes (0.25 μM) were formed in 40 μl of assay buffer solution as described in the Material and methods section. (A) Various amounts of EcoEF-G (0.05, 0.125, 0.25, 0.5, 0.75 and 1.0 μM) and GTP (0.5 mM) were then added and incubated at room temperature for 15 min. The mixture was subjected to Microcon-100 ultrafiltration to separate the ribosomes from the released RRFs. The remaining ribosome-bound RRFs were determined by quantitative Western blotting. The RRFs bound to ribosomes in the absence of EF-Gs was taken as 100%. (B) Various amounts of TteEF-G (0.05, 0.125, 0.25, 0.5, 0.75 and 1.0 μM) and GTP (0.5 mM) were applied to the assay system. Others are the same as in (A).

Figure 5. Binding of RRFs to 70 S E. coli ribosomes under different conditions

(A) The dose-response curve for binding of various amounts of EcoRRF, EcoDI/TteDII, TteDI/EcoDII or TteRRF to ribosomes in the absence of EF-Gs and GDPNP. The concentration of 70 S ribosomes is 0.25 μM. The RRF concentrations were from 0–4 μM. (B) The left column shows the binding curve of EcoRRF, EcoDI/TteDII, TteDI/EcoDII or TteRRF to EcoEF-G–ribosome complexes without (top) or with (bottom) 3.0 μM GDPNP; the right column shows the binding curve of EcoRRF, EcoDI/TteDII, TteDI/EcoDII or TteRRF to TteEF-G–ribosome complexes without (top) or with (bottom) 3.0 μM GDPNP. The concentrations of EcoEF-G and TteEF-G were 1 μM respectively. Other conditions were similar to those in (A).