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. 2018 Jun 11;7:e34252. doi: 10.7554/eLife.34252

Figure 6. The role of GTP hydrolysis for RF1 and RF3 recycling.

(A,B) Effect of different nucleotides on peptidyl-tRNA hydrolysis (GTP, black circles; GTPγS, green circles; GDPNP, blue circles; GDP, red circles; no nucleotide, grey circles) or in the presence of RF3(H92A) and GTP (purple circles). Control experiments are in the absence of RF3 (blue crosses). Error bars represent the range of two technical replicates. (A) Peptide hydrolysis was performed by incubating PreHC (100 nM) with RF3 (10 nM) and the respective nucleotides (1 mM); reactions were started with the addition of RF1 (10 nM). (B) Same as in (A), but at 1 µM RF3. (C,D) FRET distribution reporting on subunit rotation of S6/L9-labeled PreHC in the presence of saturating amounts of RF3–GDPNP (C) or RF1 with RF3–GDPNP (D) (1 µM RF each). Red lines represent the distribution of FRET states with RF3–GTP. (E,F) Contour plots representing the residence time of RF3-Cy5 (10 nM) on PostHC labeled at L11 by Cy3 in the presence of GDPNP (1 mM) without RF1 (E) or (F) in the presence of saturating RF1 concentration (1 µM). FRET values (mean ± sd) center at 0.71 ± 0.01 and 0.40 ± 0.01 (E) and 0.58 ± 0.03 (F). All values are mean ± sd from three independent data sets. See also Figure 6—figure supplement 1, Figure 6—figure supplement 2 and Supplementary file 1.

Figure 6.

Figure 6—figure supplement 1. Effect of the nucleotide bound to RF3 on subunit rotation.

Figure 6—figure supplement 1.

FRET distribution in the S6/L9-labeled PreHC in the presence of. (A) RF3 and GDP. (B) apo-RF3 in the absence of added nucleotide. (C) RF1, RF3 and GDP. (D) RF1 and apo-RF3. (E) RF3(H92A) and GTP. (F) RF3 and GTPγS. (G) RF1, RF3 and GTPγS. The concentration of RF1 and RF3 is 1 µM and of the nucleotide 1 mM. Grey lines represent the distribution of FRET states in the absence of RF3.
Figure 6—figure supplement 2. Dissociation of RF1 from PostHC mediated by RF3 in the absence of GTP hydrolysis.

Figure 6—figure supplement 2.

(A,B) In the presence of GDPNP. (C,D) In the presence of GTPγS. (E,F) With RF3(H92A) and GTP. (A,C,E) Dissociation of RF1 monitored by FRET between RF1-Cy5 (10 nM) and L11-Cy3 in the presence of excess RF3 (1 µM). The nucleotide concentration is 1 mM. Time courses were synchronized to the onset of the FRET event and combined in contour plots. FRET values (mean ±sd, from three independent data sets) are 0.47 ± 0.04 and 0.67 ± 0.04 (A), 0.66 ± 0.04 (C), and 0.51 ± 0.03 (E). (B,D,F) Dwell time distributions and the rates of RF1 and RF3 dissociation and subunit rotation.