The role of GTP hydrolysis by EF-G in ribosomal translocation

Abstract

Translocation of transfer RNA (tRNA) and messenger RNA (mRNA) through the ribosome is catalyzed by the GTPase elongation factor G (EF-G) in bacteria. Although guanosine-5'-triphosphate (GTP) hydrolysis accelerates translocation and is required for dissociation of EF-G, its fundamental role remains unclear. Here, we used ensemble Förster resonance energy transfer (FRET) to monitor how inhibition of GTP hydrolysis impacts the structural dynamics of the ribosome. We used FRET pairs S12-S19 and S11-S13, which unambiguously report on rotation of the 30S head domain, and the S6-L9 pair, which measures intersubunit rotation. Our results show that, in addition to slowing reverse intersubunit rotation, as shown previously, blocking GTP hydrolysis slows forward head rotation. Surprisingly, blocking GTP hydrolysis completely abolishes reverse head rotation. We find that the S13-L33 FRET pair, which has been used in previous studies to monitor head rotation, appears to report almost exclusively on intersubunit rotation. Furthermore, we find that the signal from quenching of 3'-terminal pyrene-labeled mRNA, which is used extensively to follow mRNA translocation, correlates most closely with reverse intersubunit rotation. To account for our finding that blocking GTP hydrolysis abolishes a rotational event that occurs after the movements of mRNA and tRNAs are essentially complete, we propose that the primary role of GTP hydrolysis is to create an irreversible step in a mechanism that prevents release of EF-G until both the tRNAs and mRNA have moved by one full codon, ensuring productive translocation and maintenance of the translational reading frame.

Keywords: FRET; frameshifting; ribosome; translation.

Fig. 1.

Positions of FRET pairs used to monitor ribosome dynamics. (A) S12-S19 and (B) S11-S13 FRET pairs report on rotation of the 30S head domain (8). (C) S6-L9 FRET pair reports on intersubunit rotation (7). Ribosomal components are 16S ribosomal RNA (rRNA) (cyan), 30S proteins (blue), 23S rRNA (gray), 5S rRNA (light blue), and 50S proteins (magenta). Positions of fluorophores are indicated by red spheres (PDB 4V9D) (41).

Fig. 2.

Effects of inhibition of GTP hydrolysis on reverse intersubunit rotation. (A) Reverse intersubunit rotation was monitored using the S6-L9 FRET pair (7). At t = 0, a pretranslocation complex was rapidly mixed with either EF-G⋅GTP (GTP), EF-G⋅GDPNP (GDPNP), EF-G⋅GDPCP (GDPCP), EF-G⋅GTPγS (GTPγS), or EF-G(H91L)⋅GTP (H91L), and changes in ensemble FRET efficiency were measured using a stopped-flow fluorometer (Materials and Methods). Curves are single-exponential functions fitted to the experimental traces (SI Appendix, Fig. S3). With the exception of GDPCP, inhibiting GTP hydrolysis had only modest effects on the rate of reverse intersubunit rotation. (B) Pseudo-first order rate constants for the traces from panel A (SI Appendix, Fig. S3 and Table 1). Error bars represent SD about the mean. AU, arbitrary unit.

Fig. 3.

Effects of inhibition of GTP hydrolysis on 30S head rotation. Forward and reverse 30S head rotation were monitored with two anticorrelated FRET pairs (8). (A) For the S12-S19 FRET pair, decreasing and increasing acceptor fluorescence indicate forward and reverse rotation, respectively. (B) For the S11-S13 FRET pair, increasing and decreasing acceptor fluorescence indicate forward and reverse rotation, respectively. Experimental traces were fitted to double-exponential functions (SI Appendix, Figs. S4 and S5 and Table 1). Insets show expanded views of the first 500 ms. AU, arbitrary unit.

Fig. 4.

Reverse head rotation is abolished in the absence of GTP hydrolysis. Apparent rate constants for (A) forward and (B) reverse head rotation were obtained from fitting the traces from Fig. 3 to double-exponential functions (SI Appendix, Figs. S4 and S5 and Table 1). Error bars represent SD about the mean.

Fig. 5.

The S13⋅L33 FRET pair likely reports primarily on reverse intersubunit rotation. (A) A pretranslocation complex containing ribosomes with the S13(Atto540Q)-L33(Alexa488) FRET pair was combined with either EF-G⋅GTP, EF-G⋅GDPNP, or EF-G(H91L)⋅GTP in a stopped-flow fluorometer. The fluorescence signal from Alexa488 was monitored over a single round of translocation. (B) The weighted average apparent rate constants (k_app) for the biphasic FRET changes from the S13-L33 FRET pair for GTP, GDPNP, and H91L conditions (SI Appendix, Table S1 and SI Methods) correlate most closely with the respective rates of reverse intersubunit rotation monitored using the S6-L9 FRET pair and differ from those measured directly for either forward or reverse head rotation (Table 1). AU, arbitrary unit.

Fig. 6.

mRNA quenching correlates most closely with reverse intersubunit rotation. (A) Translocation was monitored by quenching of a fluorophore attached to the 3′ end (position +9) of a 24-nt mRNA (40). The biphasic experimental traces (SI Appendix, Fig. S7) were fit to double-exponential curves. (B) Weighted average apparent rate constants (k_ave) for mRNA quenching (SI Appendix, SI Methods) correspond most closely to those for reverse intersubunit rotation (Table 1 and SI Appendix, Table S2). AU, arbitrary unit.

Fig. 7.

Effects of blocking GTP hydrolysis on events during translocation. Blocking GTP hydrolysis specifically abolishes reverse rotation of the 30S subunit head domain (Figs. 3 and 4), likely due to inhibition of release of EF-G, which requires release of P_i (–15). Arrows indicate proposed order of events during a single round of translocation. Red octagon symbol indicates blockage of GTP hydrolysis by GDPNP, GDPCP, or EF-G(H91L). Red X marks indicate steps abolished by blockage of GTP hydrolysis.