Kinetically competent intermediates in the translocation step of protein synthesis

Abstract

Translocation requires large-scale movements of ribosome-bound tRNAs. Using tRNAs that are proflavin labeled and single-turnover rapid kinetics assays, we identify one or possibly two kinetically competent intermediates in translocation. EF-G.GTP binding to the pretranslocation (PRE) complex and GTP hydrolysis are rapidly followed by formation of the securely identified intermediate complex (INT), which is more slowly converted to the posttranslocation (POST) complex. Peptidyl tRNA within the INT complex occupies a hybrid site, which has a puromycin reactivity intermediate between those of the PRE and POST complexes. Thiostrepton and viomycin inhibit INT formation, whereas spectinomycin selectively inhibits INT disappearance. The effects of other translocation modulators suggest that EF-G-dependent GTP hydrolysis is more important for INT complex formation than for INT complex conversion to POST complex and that subtle changes in tRNA structure influence coupling of tRNA movement to EF-G.GTP-induced conformational changes.

Figure 1

EF-G.GTP dependent translocation. (A) As measured by fMetPhe-puromycin formation. k_app values are (in s^-1): Filled circles, unlabeled tRNAs, 1.9 ± 0.1 ; open circles, tRNA^fMet(prf20), 1.5 ± 0.1; squares, tRNA^Phe(prf16/20), 1.6 ± 0.1; triangles, tRNA^Phe(prf16/17), 1.8 ± 0.2 s⁻¹. (B) – (D) As measured by changes in fluorescence of prf-labeled tRNAs as a function of EF-G concentration, in μM (green, 0.5; red, 1.0; blue, 2.0; yellow, 3.0; magenta, 5.0). (B) using yeast fMetPhe-tRNA^Phe(prf16/17) (PRE 16/17A). (C) Using E. coli fMetPhe-tRNA^Phe(prf16/20) (PRE 16/20A). (D) Using E. coli prf-tRNA^fMet(prf20) (PRE 20P). Traces shown were obtained with E. coli fMetPhe-tRNA^Phe - results obtained with yeast fMetPhe-tRNA^Phe were superimposable. Curves were fit with the k_cat and K_m(EF-G) values given in Table 1, and the following relative fluorescent values (PRE complex equals 1.0): PRE 16/17A POST 1.09; PRE 16/20A INT 1.19, POST 1.13; PRE 20P INT 1.054, POST 0.90.

Figure 1

EF-G.GTP dependent translocation. (A) As measured by fMetPhe-puromycin formation. k_app values are (in s^-1): Filled circles, unlabeled tRNAs, 1.9 ± 0.1 ; open circles, tRNA^fMet(prf20), 1.5 ± 0.1; squares, tRNA^Phe(prf16/20), 1.6 ± 0.1; triangles, tRNA^Phe(prf16/17), 1.8 ± 0.2 s⁻¹. (B) – (D) As measured by changes in fluorescence of prf-labeled tRNAs as a function of EF-G concentration, in μM (green, 0.5; red, 1.0; blue, 2.0; yellow, 3.0; magenta, 5.0). (B) using yeast fMetPhe-tRNA^Phe(prf16/17) (PRE 16/17A). (C) Using E. coli fMetPhe-tRNA^Phe(prf16/20) (PRE 16/20A). (D) Using E. coli prf-tRNA^fMet(prf20) (PRE 20P). Traces shown were obtained with E. coli fMetPhe-tRNA^Phe - results obtained with yeast fMetPhe-tRNA^Phe were superimposable. Curves were fit with the k_cat and K_m(EF-G) values given in Table 1, and the following relative fluorescent values (PRE complex equals 1.0): PRE 16/17A POST 1.09; PRE 16/20A INT 1.19, POST 1.13; PRE 20P INT 1.054, POST 0.90.

Figure 1

EF-G.GTP dependent translocation. (A) As measured by fMetPhe-puromycin formation. k_app values are (in s^-1): Filled circles, unlabeled tRNAs, 1.9 ± 0.1 ; open circles, tRNA^fMet(prf20), 1.5 ± 0.1; squares, tRNA^Phe(prf16/20), 1.6 ± 0.1; triangles, tRNA^Phe(prf16/17), 1.8 ± 0.2 s⁻¹. (B) – (D) As measured by changes in fluorescence of prf-labeled tRNAs as a function of EF-G concentration, in μM (green, 0.5; red, 1.0; blue, 2.0; yellow, 3.0; magenta, 5.0). (B) using yeast fMetPhe-tRNA^Phe(prf16/17) (PRE 16/17A). (C) Using E. coli fMetPhe-tRNA^Phe(prf16/20) (PRE 16/20A). (D) Using E. coli prf-tRNA^fMet(prf20) (PRE 20P). Traces shown were obtained with E. coli fMetPhe-tRNA^Phe - results obtained with yeast fMetPhe-tRNA^Phe were superimposable. Curves were fit with the k_cat and K_m(EF-G) values given in Table 1, and the following relative fluorescent values (PRE complex equals 1.0): PRE 16/17A POST 1.09; PRE 16/20A INT 1.19, POST 1.13; PRE 20P INT 1.054, POST 0.90.

Figure 1

EF-G.GTP dependent translocation. (A) As measured by fMetPhe-puromycin formation. k_app values are (in s^-1): Filled circles, unlabeled tRNAs, 1.9 ± 0.1 ; open circles, tRNA^fMet(prf20), 1.5 ± 0.1; squares, tRNA^Phe(prf16/20), 1.6 ± 0.1; triangles, tRNA^Phe(prf16/17), 1.8 ± 0.2 s⁻¹. (B) – (D) As measured by changes in fluorescence of prf-labeled tRNAs as a function of EF-G concentration, in μM (green, 0.5; red, 1.0; blue, 2.0; yellow, 3.0; magenta, 5.0). (B) using yeast fMetPhe-tRNA^Phe(prf16/17) (PRE 16/17A). (C) Using E. coli fMetPhe-tRNA^Phe(prf16/20) (PRE 16/20A). (D) Using E. coli prf-tRNA^fMet(prf20) (PRE 20P). Traces shown were obtained with E. coli fMetPhe-tRNA^Phe - results obtained with yeast fMetPhe-tRNA^Phe were superimposable. Curves were fit with the k_cat and K_m(EF-G) values given in Table 1, and the following relative fluorescent values (PRE complex equals 1.0): PRE 16/17A POST 1.09; PRE 16/20A INT 1.19, POST 1.13; PRE 20P INT 1.054, POST 0.90.

Figure 2

Antibiotic effects on rates of fluorescent tRNA translocation. EF-G.GTP (1 μM) was rapidly mixed with the following PRE complexes. (A) PRE 16/17A. (B) PRE 16/20A. (C) PRE 20P. Antibiotic concentrations: Spc, 1 mM; ThS, 5 μM; Vio, 5 μM. All concentrations are final. Calculated rate constants (s⁻¹) : + Spc: (A) 9.5 ± 0.4, (B) 13 ± 1, (C) 9.2 ± 0.7; + Vio: (C) 8.9 ± 0.8.

Figure 2

Antibiotic effects on rates of fluorescent tRNA translocation. EF-G.GTP (1 μM) was rapidly mixed with the following PRE complexes. (A) PRE 16/17A. (B) PRE 16/20A. (C) PRE 20P. Antibiotic concentrations: Spc, 1 mM; ThS, 5 μM; Vio, 5 μM. All concentrations are final. Calculated rate constants (s⁻¹) : + Spc: (A) 9.5 ± 0.4, (B) 13 ± 1, (C) 9.2 ± 0.7; + Vio: (C) 8.9 ± 0.8.

Figure 2

Antibiotic effects on rates of fluorescent tRNA translocation. EF-G.GTP (1 μM) was rapidly mixed with the following PRE complexes. (A) PRE 16/17A. (B) PRE 16/20A. (C) PRE 20P. Antibiotic concentrations: Spc, 1 mM; ThS, 5 μM; Vio, 5 μM. All concentrations are final. Calculated rate constants (s⁻¹) : + Spc: (A) 9.5 ± 0.4, (B) 13 ± 1, (C) 9.2 ± 0.7; + Vio: (C) 8.9 ± 0.8.

Figure 3

GTP analogue and tRNA mutation effects on rates of fluorescent tRNA translocation. All concentrations are final. G nucleotide concentrations were 0.5 mM except as otherwise indicated. PRE complexes were 0.1 μM. Translocation was initiated by rapid mixing of EF-G.G nucleotide with PRE complex. (A) Translocation of PRE 20P complex, prepared using two sucrose cushion centrifugation steps, in the presence of GTP (0.05 mM, green; 0.5 mM red), GDPNP (0.5 mM purple), or GTP-free GDP (0.05 mM black; 0.5 mM blue) or in the absence of added G-nucleotide (yellow). EF-G is 1 μM. An essentially identical final fluorescence value was obtained for the traces in the presence of GTP or GDPNP. Inset compares early time period in the presence of 0.5 mM GTP (red) or GDPNP (purple). (B) Translocation of PRE 20P complex as a function of EF-G.GDPNP concentration (in μM) (green, 0.5; red, 1; blue, 3; magenta, 5). The light blue curve is for a sample containing 5 μM EF-G.GDPNP and 4 mM Spc. (C) SDS-PAGE analysis of proteins extracted from unlabeled PRE complex incubated with EF-G and the nucleotides indicated. FA is fusidic acid. Only the region of the gel corresponding to ribosomal protein S1 and EF-G is shown. (D) EF-G.GTP dependent translocation of PRE (16/20) complex using the following forms of tRNA^fMet: native, wt-transcript, G18A variant, or U55A variant. EF-G is 2 μM.

Figure 3

GTP analogue and tRNA mutation effects on rates of fluorescent tRNA translocation. All concentrations are final. G nucleotide concentrations were 0.5 mM except as otherwise indicated. PRE complexes were 0.1 μM. Translocation was initiated by rapid mixing of EF-G.G nucleotide with PRE complex. (A) Translocation of PRE 20P complex, prepared using two sucrose cushion centrifugation steps, in the presence of GTP (0.05 mM, green; 0.5 mM red), GDPNP (0.5 mM purple), or GTP-free GDP (0.05 mM black; 0.5 mM blue) or in the absence of added G-nucleotide (yellow). EF-G is 1 μM. An essentially identical final fluorescence value was obtained for the traces in the presence of GTP or GDPNP. Inset compares early time period in the presence of 0.5 mM GTP (red) or GDPNP (purple). (B) Translocation of PRE 20P complex as a function of EF-G.GDPNP concentration (in μM) (green, 0.5; red, 1; blue, 3; magenta, 5). The light blue curve is for a sample containing 5 μM EF-G.GDPNP and 4 mM Spc. (C) SDS-PAGE analysis of proteins extracted from unlabeled PRE complex incubated with EF-G and the nucleotides indicated. FA is fusidic acid. Only the region of the gel corresponding to ribosomal protein S1 and EF-G is shown. (D) EF-G.GTP dependent translocation of PRE (16/20) complex using the following forms of tRNA^fMet: native, wt-transcript, G18A variant, or U55A variant. EF-G is 2 μM.

Figure 3

GTP analogue and tRNA mutation effects on rates of fluorescent tRNA translocation. All concentrations are final. G nucleotide concentrations were 0.5 mM except as otherwise indicated. PRE complexes were 0.1 μM. Translocation was initiated by rapid mixing of EF-G.G nucleotide with PRE complex. (A) Translocation of PRE 20P complex, prepared using two sucrose cushion centrifugation steps, in the presence of GTP (0.05 mM, green; 0.5 mM red), GDPNP (0.5 mM purple), or GTP-free GDP (0.05 mM black; 0.5 mM blue) or in the absence of added G-nucleotide (yellow). EF-G is 1 μM. An essentially identical final fluorescence value was obtained for the traces in the presence of GTP or GDPNP. Inset compares early time period in the presence of 0.5 mM GTP (red) or GDPNP (purple). (B) Translocation of PRE 20P complex as a function of EF-G.GDPNP concentration (in μM) (green, 0.5; red, 1; blue, 3; magenta, 5). The light blue curve is for a sample containing 5 μM EF-G.GDPNP and 4 mM Spc. (C) SDS-PAGE analysis of proteins extracted from unlabeled PRE complex incubated with EF-G and the nucleotides indicated. FA is fusidic acid. Only the region of the gel corresponding to ribosomal protein S1 and EF-G is shown. (D) EF-G.GTP dependent translocation of PRE (16/20) complex using the following forms of tRNA^fMet: native, wt-transcript, G18A variant, or U55A variant. EF-G is 2 μM.

Figure 3

GTP analogue and tRNA mutation effects on rates of fluorescent tRNA translocation. All concentrations are final. G nucleotide concentrations were 0.5 mM except as otherwise indicated. PRE complexes were 0.1 μM. Translocation was initiated by rapid mixing of EF-G.G nucleotide with PRE complex. (A) Translocation of PRE 20P complex, prepared using two sucrose cushion centrifugation steps, in the presence of GTP (0.05 mM, green; 0.5 mM red), GDPNP (0.5 mM purple), or GTP-free GDP (0.05 mM black; 0.5 mM blue) or in the absence of added G-nucleotide (yellow). EF-G is 1 μM. An essentially identical final fluorescence value was obtained for the traces in the presence of GTP or GDPNP. Inset compares early time period in the presence of 0.5 mM GTP (red) or GDPNP (purple). (B) Translocation of PRE 20P complex as a function of EF-G.GDPNP concentration (in μM) (green, 0.5; red, 1; blue, 3; magenta, 5). The light blue curve is for a sample containing 5 μM EF-G.GDPNP and 4 mM Spc. (C) SDS-PAGE analysis of proteins extracted from unlabeled PRE complex incubated with EF-G and the nucleotides indicated. FA is fusidic acid. Only the region of the gel corresponding to ribosomal protein S1 and EF-G is shown. (D) EF-G.GTP dependent translocation of PRE (16/20) complex using the following forms of tRNA^fMet: native, wt-transcript, G18A variant, or U55A variant. EF-G is 2 μM.

Figure 4

Mechanism of translocation showing stepwise movement of tRNA following GTP hydrolysis. Two new intermediates are shown in blue. Inhibitions by antibiotics or tRNA mutations are shown by red dashed lines.

Scheme 1