Interrupted catalysis: the EF4 (LepA) effect on back-translocation

Abstract

EF4, although structurally similar to the translocase EF-G, promotes back-translocation of tRNAs on the ribosome and is important for bacterial growth under certain conditions. Here, using a coordinated set of in vitro kinetic measures, including changes in the puromycin reactivity of peptidyl-tRNA and in the fluorescence of labeled tRNAs and mRNA, we elucidate the kinetic mechanism of EF4-catalyzed back-translocation and determine the effects of the translocation inhibitors spectinomycin and viomycin on the process. EF4-dependent back-translocation proceeds from a post-translocation (POST) complex to a pre-translocation (PRE) complex via a four-step kinetic scheme (i.e., POST-->I(1)-->I(2)-->I(3)-->PRE, which is not the simple reverse of translocation). During back-translocation, movements of the tRNA core regions and of mRNA are closely coupled to one another but are sometimes decoupled from movement of the 3'-end of peptidyl-tRNA. EF4 may be thought of as performing an interrupted catalysis of back-translocation, stopping at the formation of I(3) rather than catalyzing the complete process of back-translocation culminating in PRE complex formation. The delay in polypeptide elongation resulting from transient accumulation of I(3) is likely to be important for optimizing functional protein biosynthesis.

Figure 1. Reactivity of peptidyl-tRNA toward puromycin and EF4-dependent GTPase

A. Isolated POST complex (0.1 μM) containing fMetPhe-tRNA^Phe in the P-site and tRNA^fMet in the E–site was incubated for the times indicated with a solution containing tRNA^fMet (0.15 μM) and either GTP or GDPNP in the presence or absence of EF4 (3 μM when present) at 25 °C. For fMetPhe-puromycin formation, the incubation period was followed by reaction for 15 s with 1 mM puromycin. () -GTP, -EF4; () GTP, 2 mM, + EF4; () GTP, 0.5 mM, + EF4; () GDPNP, 0.5 mM + EF4. For GTP hydrolysis (■), the incubations were carried out with EF4 and γ-³²P GTP (50 μM) followed directly by quenching. Concentrations are after mixing. B. fMetPhe-puromycin formation was carried out as in A. with varying concentrations of EF4, except that 2 mM puromycin was employed.

Figure 2. EF4-dependent back translocation measured by change in the fluorescence of fMetPhe-tRNA^Phe(prf) and tRNA^fMet(prf)

Fluorescence changes measured in a stopped-flow spectrofluorimeter over different time scales. The dashed lines through the red traces are the results of fits to Scheme 1 (Figure 5). A and B. fMetPhe-tRNA^Phe(prf) (P-site) fluorescence change over different time scales. POST complex (0.1 μM) containing fMetPhe-tRNA^Phe(Prf) in the P-site and tRNA^fMet in the E–site was rapidly mixed with 0.15 μM tRNA^fMet and either 3 μM EF4•GDPNP (red trace), or 3 μM EF4•GDP (orange trace); or 3 μM EF4•GDPNP and either 1 mM Spc (black trace) or 1 mM Vio (dark blue trace); or just 1 mM Spc alone (light green trace), or 1 mM Vio alone (pink trace), or GDPNP alone (teal trace). C and D. tRNA^fMet(prf) (E-site) fluorescence change over different time scales. POST complexes (0.1 μM) containing fMetPhe-tRNA^Phe in the P-site and tRNA^fMet(prf) in the E–site were rapidly mixed with 0.15 μM tRNA^fMet(prf) and 3 μM EF4•GDPNP in the absence (red) or presence (black) of 1 mM Spc. The small but rapid rise in fluorescence occurring immediately after mixing that is evident in parts B and D is likely due to EF4 binding (data not shown). It is complete within 0.15 s and may be considered as occurring instantaneously with respect to the subsequent reaction phases described in the text.

Figure 3. EF-G-dependent translocation and EF4-dependent back translocation measured by change in the fluorescence of Flu-mRNA014

Fluorescence changes measured in a stopped-flow spectrofluorometer. Ribosomes were programmed with Flu-mRNA14 except as otherwise indicated. The dashed lines through traces are the results of fits to Scheme 1 (Figure 5). A. Translocation. PRE complexes (0.1 μM) were rapidly mixed with 3 μM EFG•GTP in either Buffer C (blue trace) or D (50 mM Tris-HCl [pH 7.5], 70 mM NH₄Cl, 30 mM KCl, 7 mM MgCl₂, and 1 mM DTT) (red trace). Buffer D was used in Pan et al (2007). B - D. Back translocation. POST complexes (0.1 μM) containing 0.15 μM added tRNA^fMet were rapidly mixed with EF4•GDPNP. B. Using varying EF4•GDPNP concentrations. C. The initial phase of back translocation, as monitored by the fluorescence change of Flu-mRNA14 (red trace), fMetPhe-tRNA^Phe(prf) (brown trace), or tRNA^fMet(prf) (blue trace). In the latter two cases, ribosomes were programmed with mRNA MFK. For ease of comparison, fluorescence changes are normalized to the total change for full back translocation. POST complex concentration was increased to 0.3 μM for Flu-mRNA14 to increase signal-to-noise ratio. Very similar results were obtained at 0.1 μM. D. The effects of added Spc (5 mM) (black trace) and Vio (1 mM) (blue trace) on EF4-dependent back translocation (no added antibiotic) (red trace).

Figure 4. EF4-dependent back translocation measured by the change in puromycin reactivity of fMetPhe-tRNA^Phe

A. Design of the preincubation-incubation experiment (adapted from a figure presented in reference 26). POST complex was rapidly mixed with EF4•GDPNP and E. coli tRNA^fMet and preincubated prior to rapid mixing with puromycin, further incubation and quenching. B. POST complex was rapidly mixed with EF4•GDPNP and E. coli tRNA^fMet and preincubated for the indicated times [0.02 s (); 3 s (); 7 s (); 65 s (); 200 s ()] prior to rapid mixing and incubation with 5 mM puromycin for up to 50 s, and quenching. The preincubation mixtures contained 0.1 μM POST complex, 3 μM EF4•GDPNP and 0.15 μM E.coli tRNA^fMet. Lines through the data are fits to Scheme 1 (Figure 5). A control sample in which preincubation was carried out for 200 s in the absence of EF4 showed no loss in puromycin reactivity ().C. As described in B, but in the presence of 5 mM spectinomycin. Preincubation times were: 1 s (); 3 s (); 65 s (); 200 s (). All traces are fit by single exponential, with an apparent rate constant of 0.30 ± 0.03 s⁻¹. D. The effect of long pre-incubation times. 0.1 μM of POST complex was preincubated for the times indicated with 0.15 μM E.coli tRNA^fMet and various other combinations [3 μM EF4•GDPNP (); 3 μM EF4•GDPNP plus 1 mM Vio (); 1 mM Vio in the absence of EF4 (▲)], prior to incubation with puromycin (5 mM) for 20 s prior to quenching. The EF4•GDPNP trace is fit to a single exponential, giving an apparent rate constant of 7.9± 0.5 × 10⁻⁴ s⁻¹. The EF4•GDPNP plus 1 mM Vio and 1 mM Vio alone traces are fit by double exponentials, giving identical values of 0.011 ± 0.005 s⁻¹ and 8.8 ± 0.9 × 10⁻⁴ s⁻¹ for each trace.

Figure 5. A quantitative kinetic scheme for interrupted EF4 catalysis of back translocation

EF4 catalyzes back translocation via steps 1 - 3, but has little effect on step 4. The rate constants shown are the results of global fitting to the results, measured at 3 μM EF4•GDPNP, in the absence of antibiotic, presented in Figures 2, 3B,C and 4B,D. Apparent rate constants for fMetPhe-puromycin formation were measured at 5 mM puromycin.