Elongation factor G stabilizes the hybrid-state conformation of the 70S ribosome

Abstract

Following peptide bond formation, transfer RNAs (tRNAs) and messenger RNA (mRNA) are translocated through the ribosome, a process catalyzed by elongation factor EF-G. Here, we have used a combination of chemical footprinting, peptidyl transferase activity assays, and mRNA toeprinting to monitor the effects of EF-G on the positions of tRNA and mRNA relative to the A, P, and E sites of the ribosome in the presence of GTP, GDP, GDPNP, and fusidic acid. Chemical footprinting experiments show that binding of EF-G in the presence of the non-hydrolyzable GTP analog GDPNP or GDP.fusidic acid induces movement of a deacylated tRNA from the classical P/P state to the hybrid P/E state. Furthermore, stabilization of the hybrid P/E state by EF-G compromises P-site codon-anticodon interaction, causing frame-shifting. A deacylated tRNA bound to the P site and a peptidyl-tRNA in the A site are completely translocated to the E and P sites, respectively, in the presence of EF-G with GTP or GDPNP but not with EF-G.GDP. Unexpectedly, translocation with EF-G.GTP leads to dissociation of deacylated tRNA from the E site, while tRNA remains bound in the presence of EF-G.GDPNP, suggesting that dissociation of tRNA from the E site is promoted by GTP hydrolysis and/or EF-G release. Our results show that binding of EF-G in the presence of GDPNP or GDP.fusidic acid stabilizes the ribosomal intermediate hybrid state, but that complete translocation is supported only by EF-G.GTP or EF-G.GDPNP.

FIGURE 1.

Chemical footprinting of a ribosome·tRNA^fMet·EF-G·GDPNP complex. Lanes A, C, and G, sequencing lanes; lane K, unmodified rRNA; lane 1, 70S ribosomes and mRNA32; lane 2, 70S ribosomes, mRNA32, and fMet-tRNA^fMet; lane 3, 70S ribosomes, mRNA32, fMet-tRNA^fMet, and puromycin; lane 4, 70S ribosomes, mRNA32, fMet-tRNA^fMet, puromycin, and EF-G·GDPNP; lane 5, 70S ribosomes, mRNA32, fMet-tRNA^fMet, puromycin, and EF-G·GTP. (A) 16S rRNA P site; (B) 23S rRNA P site; (C) 23S rRNA E site; (D) 16S rRNA inter-subunit bridge B7a; (E) 23S rRNA sarcin–ricin loop (SRL, EF-G binding site); (F) schematic indicating movement of tRNA^fMet in the presence of EF-G·GDPNP.

FIGURE 2.

Chemical footprinting of a ribosome·tRNA^Phe·EF-G·GDPNP complex. Lanes A, C, and G, sequencing lanes; lane K, unmodified rRNA; lane 1, 70S ribosomes and mRNA32; lane 2, 70S ribosomes, mRNA32, and N-Ac-Phe-tRNA^Phe; lane 3, 70S ribosomes, mRNA32, N-Ac-Phe-tRNA^Phe, and puromycin; lane 4, 70S ribosomes, mRNA32, N-Ac-Phe-tRNA^Phe, puromycin, and EF-G·GDPNP; lane 5, 70S ribosomes, mRNA32, N-Ac-Phe-tRNA^Phe, puromycin, and EF-G·GTP. (A) 16S rRNA P site; (B) 23S rRNA P site; (C) 23S rRNA E site; (D) 16S rRNA inter-subunit bridge B7a; (E) 23S rRNA sarcin–ricin loop (SRL, EF-G binding site); (F) schematic indicating movement of tRNA^Phe in the presence of EF-G·GDPNP.

FIGURE 3.

Chemical footprinting of a ribosome·tRNA^Phe·EF-G·(GTP/GDP)·fusidic acid complex. Lanes A, C, and G, sequencing lanes; lane K, unmodified rRNA; lane 1, 70S ribosomes and mRNA32; lane 2, 70S ribosomes, mRNA32, and N-Ac-Phe-tRNA^Phe, and puromycin; lane 3, 70S ribosomes, mRNA32, N-Ac-Phe-tRNA^Phe, puromycin, and EF-G·GTP; lane 4, 70S ribosomes, mRNA32, N-Ac-Phe-tRNA^Phe, puromycin, EF-G·GTP, and fusidic acid; lane 5, 70S ribosomes, mRNA32, N-Ac-Phe-tRNA^Phe, puromycin, and EF-G·GDPNP; lane 6, 70S ribosomes, mRNA32, N-Ac-Phe-tRNA^Phe, puromycin, and EF-G·GDP; lane 7, 70S ribosomes, mRNA32, N-Ac-Phe-tRNA^Phe, puromycin, EF-G·GDP, and fusidic acid. (A) 16S rRNA P site; (B) 23S rRNA P site; (C) 23S rRNA E site; (D) 16S rRNA inter-subunit bridge B7a; (E) 23S rRNA sarcin–ricin loop (SRL, EF-G binding site); (F) 23S rRNA GTPase-associated center (GAC, EF-G binding site); (G) schematic indicating movement of tRNA^Phe in the presence of EF-G·(GTP or GDP)·fusidic acid.

FIGURE 4.

Interaction of EF-G with the ribosome is required for mRNA back-slippage. (A) A pre-translocation complex was made by binding deacylated tRNA^Tyr to the P site and N-Ac-Phe-tRNA^Phe to the A site in the presence of mRNA 301. Some of the pre-translocation complexes were incubated with EF-G and GTP to allow translocation to proceed. Newly formed post-translocation complexes were separated into three aliquots. The first aliquot was left unperturbed, the third was initially incubated with thiostrepton to disrupt the interaction of EF-G with the ribosome, and the second and third aliquots were each incubated with puromycin to deacylate the P-site–bound N-Ac-Phe-tRNA^Phe as indicated. The position of the ribosome along the mRNA was mapped by toeprinting. A toeprint band at +16 corresponds to pre-translocation complex, a band at +19 is the product of accurate translocation, and a band at +14 is the product of mRNA back-slippage. (B) Schematic illustrating mechanism of back-slippage. Incubation of the ribosome with puromycin after translocation results in a deacylated tRNA^Phe bound in the P site. Binding of EF-G to the ribosome induces a movement of tRNA^Phe from the classical P/P state to the hybrid P/E state, which destabilizes tRNA·mRNA interactions and results in mRNA back-slippage. Thiostrepton disrupts interaction of EF-G with ribosome and prevents mRNA back-slippage.

FIGURE 5.

EF-G·GDPNP is efficient in promoting mRNA back-slippage. (A) A pre-translocation complex was made by binding deacylated tRNA^Tyr to the P site and deacylated tRNA^Phe to the A site in the presence of mRNA 301. Some of the pre-translocation complexes were incubated with EF-G·GTP or EF-G·GDPNP as indicated. The position of the ribosome along the mRNA was mapped by toeprinting. A toeprint band at +16 corresponds to the pre-translocation complex, a band at +19 is the product of accurate translocation, and a band at +14 is the product of mRNA back-slippage. (B) Schematic illustrating the mechanism of mRNA back-slippage. When deacylated tRNA^Phe is translocated from the A site to the P site, it moves from the classical P/P state to the hybrid P/E state as EF-G binds to the ribosome. Hybrid-state formation results in a destabilization of the codon–anticodon interactions and results in mRNA back slippage. EF-G·GDPNP binds to the ribosome more stably and promotes mRNA back-slippage more efficiently than EF-G·GTP.

FIGURE 6.

Puromycin reactivity of pre-translocation state ribosome complexes incubated with EF-G. (A) Diagram indicating the puromycin reactivity of the pre- and post-translocation state ribosome complexes. Deacylated tRNA^fMet was bound to the 30S P site, and N-Ac-[¹⁴C]Phe-tRNA^Phe was bound to the 30S A site. (B) Histograms indicate the relative amount of N-Ac-[¹⁴C]Phe-tRNA^Phe that is bound to the ribosome. (C) Histograms indicate the fraction of ribosome-bound N-Ac-[¹⁴C]Phe-tRNA^Phe that is puromycin reactive: (pre-trans) the pre-translocation complex as described in A; (GDPNP) non-hydrolyzable GTP analog; (fus) fusidic acid.

FIGURE 7.

Chemical footprinting of the pre-translocation complex in the presence of EF-G·GDPNP. Lanes A, C, and G, sequencing lanes; lane K, unmodified rRNA; lane 1, 70S ribosomes and mRNA32; lane 2, 70S ribosomes, mRNA32, and fMet-tRNA^fMet; lane 3, 70S ribosomes, mRNA32, fMet-tRNA^fMet, and puromycin; lane 4, 70S ribosomes, mRNA32, fMet-tRNA^fMet, puromycin, and N-Ac-Phe-tRNA^Phe; lane 5, 70S ribosomes, mRNA32, fMet-tRNA^fMet, puromycin, N-Ac-Phe-tRNA^Phe, and EF-G·GDPNP; lane 6, 70S ribosomes, mRNA32, fMet-tRNA^fMet, puromycin, N-Ac-Phe-tRNA^Phe, and EF-G·GTP. (A) 16S rRNA P site; (B) 23S rRNA P site; (C) 16S rRNA A site; (D) 23S rRNA E site; (E) 23S rRNA sarcin–ricin loop (SRL, EF-G binding site); (F) schematic indicating movement of deacylated tRNA^fMet (P site) and N-Ac-Phe-tRNA^Phe (A site) in the presence of EF-G·GDPNP.

FIGURE 8.

Toeprinting analysis of mRNA translocation catalyzed by EF-G in the presence of different nucleotides and antibiotics. The pre-translocation complex was assembled by binding deacylated tRNA^Met (P site), N-Ac-Phe-tRNA^Phe (A site), and m291 mRNA to the ribosome. The pre-translocation complex was incubated with EF-G in the presence of GTP, GDPNP, GDP, and fusidic acid as indicated. The toeprint bands at +16 and +19 correspond to the pre- and post-translocation states of the ribosome, respectively.