Fluctuations between multiple EF-G-induced chimeric tRNA states during translocation on the ribosome

Abstract

The coupled translocation of transfer RNA and messenger RNA through the ribosome entails large-scale structural rearrangements, including step-wise movements of the tRNAs. Recent structural work has visualized intermediates of translocation induced by elongation factor G (EF-G) with tRNAs trapped in chimeric states with respect to 30S and 50S ribosomal subunits. The functional role of the chimeric states is not known. Here we follow the formation of translocation intermediates by single-molecule fluorescence resonance energy transfer. Using EF-G mutants, a non-hydrolysable GTP analogue, and fusidic acid, we interfere with either translocation or EF-G release from the ribosome and identify several rapidly interconverting chimeric tRNA states on the reaction pathway. EF-G engagement prevents backward transitions early in translocation and increases the fraction of ribosomes that rapidly fluctuate between hybrid, chimeric and posttranslocation states. Thus, the engagement of EF-G alters the energetics of translocation towards a flat energy landscape, thereby promoting forward tRNA movement.

Dynamic relationships between ribosomal conformational and RNA positional changes during ribosomal translocation

“…The positional change of the peptidyl-tRNA arising from the transition from State H to State H1 also causes the decrease of FRET between Cy3-labeled ribosomal protein L11 and Cy5-labeled peptidyl-tRNA (L11-t FRET) (see Fig. 1), which is consistent with the smFRET data of Adio et al [30]. The characteristic of 30S head rotations in Fig.…”

“…In summary, our proposed pathway of ribosomal translocation is consistent with diverse single molecule and ensemble kinetic data presented in the literature [15, 21, 23, 27, 30, 31, 32, 33, 34, 35, 37]. With the pathway we study the dynamic relations among different transitions or events in the translocation pathway exhibited in the smFRET data and those exhibited in the ensemble kinetic data.…”

“…S1) that is similar to that of Fig. 1, we provided quantitative explanations of diverse single molecule and ensemble kinetic data obtained by different research groups in the literature, such as (i) the smFRET data on dynamic fluctuations between different tRNA states during translocation interfered with various EF-G mutants, the antibiotic fusidic acid and non-hydrolysable GTP analogue $\text{GTP}\gamma \text{S}$ [30], (ii) the ensemble kinetic data on 30S head rotation catalyzed by EF-G hydrolyzing GTP [27], (iii) the ensemble kinetic data on tRNA movement in 50S subunit and mRNA movement in 30S subunit catalyzed by wild-type EF-G, different EF-G mutants and antibiotics [31], (iv) the ensemble kinetic data on biphasic characteristics of mRNA translocation in wild-type ribosome and in mutant ribosomes C2394A, C2394 G and C2394U, where nucleotide C2394 of 23S rRNA in the 50S subunit was mutated to A, G and U, respectively, so that the affinity of deacylated tRNA for the 50S E site was reduced greatly [32], (v) the single molecule data on the dynamics of EF-G sampling to the ribosome during ribosomal translocation [33], etc. Various ensemble kinetic data on sparsomycin-catalyzed ribosomal translocation [34, 35] were also explained well with the similar pathway [36].…”

Elongation factor G initiates translocation through a power stroke

“…Brackets are used to mark short-lived intermediates during normal translocation, similar to states captured by timeresolved cryo-EM along the translocation pathway (9). In the hybrid model, these intermediates have similar free energies in the absence of EF-G; they can spontaneously interconvert until their thermally driven fluctuations are blocked by conformational changes within EF-G that complete translocation (2). Both of these models include a power stroke early in translocation.…”

“…Translocation follows, and EF-G·GDP dissociates from the resulting POST-translocation complex (1). Recent studies (2)(3)(4) have clearly demonstrated that EF-G·GTP promotes translocation from either the classic or hybrid PRE complex, which contain tRNAs in the A/A and P/P or A/P and P/E sites, respectively (first letter 30S position, second letter 50S position).…”