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Complementary roles of initiation factor 1 and ribosome recycling factor in 70S ribosome splitting

2008; Springer Nature; Volume: 27; Issue: 12 Linguagem: Inglês

10.1038/emboj.2008.99

1460-2075

Michael Y. Pavlov, Ayman Antoun, Martin Lovmar, Måns Ehrenberg,

Peptidase Inhibition and Analysis

Article22 May 2008free access Complementary roles of initiation factor 1 and ribosome recycling factor in 70S ribosome splitting Michael Y Pavlov Michael Y Pavlov Department of Cell and Molecular Biology, BMC, Uppsala University, Uppsala, Sweden Search for more papers by this author Ayman Antoun Ayman Antoun Department of Cell and Molecular Biology, BMC, Uppsala University, Uppsala, SwedenPresent Address: Institute for Cancer Studies, University of Birmingham, Birmingham, UK Search for more papers by this author Martin Lovmar Martin Lovmar Department of Cell and Molecular Biology, BMC, Uppsala University, Uppsala, SwedenPresent Address: CMB/Microbiology, University of Gothenburg, Gothenburg, Sweden Search for more papers by this author Måns Ehrenberg Corresponding Author Måns Ehrenberg Department of Cell and Molecular Biology, BMC, Uppsala University, Uppsala, Sweden Search for more papers by this author Michael Y Pavlov Michael Y Pavlov Department of Cell and Molecular Biology, BMC, Uppsala University, Uppsala, Sweden Search for more papers by this author Ayman Antoun Ayman Antoun Department of Cell and Molecular Biology, BMC, Uppsala University, Uppsala, SwedenPresent Address: Institute for Cancer Studies, University of Birmingham, Birmingham, UK Search for more papers by this author Martin Lovmar Martin Lovmar Department of Cell and Molecular Biology, BMC, Uppsala University, Uppsala, SwedenPresent Address: CMB/Microbiology, University of Gothenburg, Gothenburg, Sweden Search for more papers by this author Måns Ehrenberg Corresponding Author Måns Ehrenberg Department of Cell and Molecular Biology, BMC, Uppsala University, Uppsala, Sweden Search for more papers by this author Author Information Michael Y Pavlov1, Ayman Antoun1, Martin Lovmar1 and Måns Ehrenberg 1 1Department of Cell and Molecular Biology, BMC, Uppsala University, Uppsala, Sweden *Corresponding author. Department of Cell and Molecular Biology, Biomedical Center, Uppsala University, Box 596, Husargatan 3, Uppsala 751 24, Sweden. Tel.:+46 18 471 4213; Fax: +46 18 471 4262; E-mail: [email protected] The EMBO Journal (2008)27:1706-1717https://doi.org/10.1038/emboj.2008.99 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info We demonstrate that ribosomes containing a messenger RNA (mRNA) with a strong Shine–Dalgarno sequence are rapidly split into subunits by initiation factors 1 (IF1) and 3 (IF3), but slowly split by ribosome recycling factor (RRF) and elongation factor G (EF-G). Post-termination-like (PTL) ribosomes containing mRNA and a P-site-bound deacylated transfer RNA (tRNA) are split very rapidly by RRF and EF-G, but extremely slowly by IF1 and IF3. Vacant ribosomes are split by RRF/EF-G much more slowly than PTL ribosomes and by IF1/IF3 much more slowly than mRNA-containing ribosomes. These observations reveal complementary splitting of different ribosomal complexes by IF1/IF3 and RRF/EF-G, and suggest the existence of two major pathways for ribosome splitting into subunits in the living cell. We show that the identity of the deacylated tRNA in the PTL ribosome strongly affects the rate by which it is split by RRF/EF-G and that IF3 is involved in the mechanism of ribosome splitting by IF1/IF3 but not by RRF/EF-G. With support from our experimental data, we discuss the principally different mechanisms of ribosome splitting by IF1/IF3 and by RRF/EF-G. Introduction When, in the bacterial cell, 70S ribosomes have finished messenger RNA (mRNA) translation, the completed proteins are released by the action of class-1 (RF1 and RF2) and class-2 (RF3) release factors (Freistroffer et al, 1997; Pavlov et al, 1997; Kisselev et al, 2003). Then, the post-termination ribosomes, still containing deacylated transfer RNA (tRNA) and mRNA, are split into subunits by ribosome recycling factor (RRF) and elongation factor G (EF-G) in a GTP-dependent reaction (Janosi et al, 1996; Karimi et al, 1999; Peske et al, 2005; Zavialov et al, 2005b), which continuously supplies the cell with large (50S) and small (30S) ribosomal subunits for new rounds of initiation of mRNA translation. Initiation of bacterial protein synthesis normally proceeds through a 30S pre-initiation complex containing, apart from the 30S subunit, an mRNA, the initiator tRNA (fMet-tRNAfMet) and the initiation factors 1 (IF1), 2 (IF2) and 3 (IF3) (Gualerzi and Pon, 1990). The first step in 30S pre-initiation complex formation after ribosome splitting by RRF and EF-G is the binding of IF3 to the 30S subunit, which induces dissociation of the deacylated tRNA and mRNA remaining from the previous round of protein synthesis (Karimi et al, 1999; Peske et al, 2005). The 30S pre-initiation complex, assembled by the subsequent binding of IF1 and IF2, a new mRNA and fMet-tRNAfMet (Benne et al, 1973; Gualerzi and Pon, 1990; Antoun et al, 2006b), forms a 70S initiation complex ready for protein elongation by rapid docking to the 50S subunit (Benne et al, 1973; Antoun et al, 2006b). Accordingly, regular splitting of the 70S ribosome into subunits during protein synthesis occurs when it is in the post-termination state with an mRNA and a deacylated tRNA in the P site. However, due to aberrant events in protein synthesis and cellular responses to various stress situations, splitting into subunits of other than post-termination ribosomes is essential for the living cell. When, for instance, ribosomes are stalled on truncated mRNAs (Singh and Varshney, 2004) or by the presence of antibiotic drugs (Tenson et al, 2003), peptidyl-tRNA drop-off (Menninger, 1976) may generate tRNA-free 70S·mRNA complexes. Such complexes may also be generated through the drop-off of peptidyl-tRNAs with short peptides at early stages of translation of specific mRNA sequences (Heurgue-Hamard et al, 2000; Gonzalez de Valdivia and Isaksson, 2005) or by premature docking of 50S subunits to 30S pre-initiation complexes lacking initiator tRNA (Antoun et al, 2006b). Furthermore, when bacteria enter stationary phase or experience cold shock, superfluous ribosomes become stored either as inactive 70S ribosomes in complex with the YfiA protein (Vila-Sanjurjo et al, 2004; Ueta et al, 2005) or as inactive 100S dimmers in complex with the RMF and YhbH proteins (Ueta et al, 2005). Such ribosomes need to be rapidly activated in response to improved growth conditions (Ueta et al, 2005). Here, we demonstrate how the addition of IF1 together with IF3 and an mRNA containing a 'strong' Shine–Dalgarno (SD) sequence increased dramatically the rate of ribosome splitting into subunits, suggesting mRNA binding followed by the combined action of IF1 and IF3 as the dominant intracellular pathway for the splitting of tRNA-less ribosomes. Ribosome splitting by RRF and EF-G was, in contrast, slowed down by the presence of an mRNA alone, but greatly accelerated by the presence of both an mRNA and a deacylated tRNA. In this latter case, the ribosomal splitting by IF1/IF3 was virtually blocked. Taken together, our data show that IF1/IF3- and RRF/EF-G-dependent splitting of different ribosomal complexes were complementary, that is, a complex rapidly split by IF1/IF3 was slowly split by RRF/EF-G and vice versa. We have also found IF3 to be involved in the mechanism of IF1/IF3-dependent but not in the mechanism of RRF/EF-G-dependent ribosome splitting, and that the splitting of post-termination-like (PTL) ribosomes by RRF/EF-G was strongly affected by the identity of the deacylated tRNA in the P site. These and other data are used to discuss the different mechanisms of ribosome splitting into subunits by IF1 and IF3 on the one hand and by RRF and EF-G on the other. Results Complementarity of IF1/IF3 and RRF/EF-G in splitting of ribosomal complexes We used an in vitro system for protein synthesis with Escherichia coli components of high activity and purity (Pavlov et al, 1997) to study the splitting of various ribosomal complexes into subunits with stopped-flow techniques in combination with monitoring of Rayleigh light scattering (Antoun et al, 2006b). The splitting reactions were normally monitored in the presence of IF3, which rapidly binds to dissociated 30S subunits and inhibits their re-association to free 50S subunits (Grunberg-Manago et al, 1975). The experiments are shown in Figure 1 and the estimated rate constants for the splitting of various ribosomal complexes are compiled in Table I. Figure 1.Splitting of 70S ribosomes and their complexes with mRNA and tRNAPhe by IF3, by IF1 and IF3 or by RRF, EF-G and IF3. The mixtures from syringe 1 of the stopped-flow instrument were rapidly mixed with equal volumes of the mixture from syringe 2 containing (A) 2 μM IF3, (B) 2 μM IF3 and 5 μM IF1 or (C) 2 μM IF3, 9 μM RRF and 4 μM EF-G. Syringe 1 contained 0.3 μM vacant 70S ribosomes (▴); 0.3 μM 70S ribosomes pre-incubated with 0.5 μM mXR7 mRNA (•), with 0.5 μM mBar mRNA (⧫), with 0.5 μM deacylated tRNAPhe (▪), with both mXR7 mRNA and tRNAPhe (▵) or 0.3 μM 70S ribosomes but with mXR7 mRNA added in syringe 2 (○). All mixtures were prepared in LS4 buffer containing 4 mM free Mg2+. All concentrations in the figure legends are given as final concentrations after the mixing. Download figure Download PowerPoint Table 1. Splitting rate q (s−1) of different ribosomal complexes in the presence of IF3, IF3 and IF1, or IF3 and RRF/EF-G IF3 IF3+IF1 IF3+RRF+EF-G 70S only 0.003±0.006 0.063±0.002 0.095±0.005 70S+tRNAPhe 0.003±0.007 0.057±0.002 0.23±0.01 70S+mXR7 0.029±0.002 0.92±0.04 0.064±0.004 70S+mBar 0.006±0.001 0.16±0.02 0.068±0.004 70S+mXR7+tRNAPhe ∼0 ∼0 1.05±0.05 70S only; mXR7 with factorsa 0.023±0.002 0.53±0.03 — The sequences of mXR7 and mBar mRNAs are shown in Table 2. All experiments were conducted at 37°C in LS4 buffer containing 4 mM Mg2+. a mXR7 mRNA was added together with IF3 or with IF1 and IF3. The rate constant, q, for the dissociation of vacant 70S ribosomes was estimated as 0.003 s−1 (Figure 1A), and remained unaltered to changes in IF3 concentration above 1 μM (data not shown). Pre-incubation of 70S ribosomes with mXR7 mRNA (mXR7) (Table II) with a strong SD sequence before addition of IF3 or simultaneous addition of IF3 and mXR7 to the vacant ribosomes increased q by about an order of magnitude to 0.029 or 0.023 s−1, respectively. When, instead, the 70S ribosomes were pre-incubated with mBar mRNA (mBar) (Table II) with a weak SD sequence before addition of IF3, there was only a two-fold increase in q (Figure 1A and Table I). Pre-incubation of the 70S ribosomes with only deacylated tRNAPhe did not alter q (data not shown), but pre-incubation with both tRNAPhe and mXR7 decreased q to virtually zero (Figure 1A). These observations suggest that, by itself, the SD–anti-SD (ASD) interaction between the mRNA leader and 16S rRNA decreased the affinity of ribosomal subunit binding, whereas this affinity was greatly increased by the simultaneous presence of an mRNA and a P-site-bound deacylated tRNA. Table 2. Complete sequences of synthetic mRNAs mRNA Complete sequence of mRNA mXR7 GggAAUUCGGGCCCUUGUUAACAAUUAAGGAGGUAUACU AUGUUUACGAUUUaaUUGCAGaaaaaaaaaaaaaaaaaaaaa mBar GggAAGCUGAACGAGAAACGUAAA AUGUUCACGAUUUaataAUCAAUAUACUGCAGaaaaaaaaaaaaaaaaaaaa mXR8 GggAAUUCGGGCCCUUGUUAACAAUUAAGGAGGUAUAUC AUGUUCACGAUCuaaUCUGCAGaaaaaaaaaaaaaaaaaaaaa mXR8_FM GggAAUUCGGGCCCUUGUUAACAAUUAAGGAGGUAUAUC UUCAUGACGAUCuaaUCUGCAGaaaaaaaaaaaaaaaaaaaaa mSD022 GggAAUUCAAAAAUUUAAAAGUUAACAGGUAUACAUACU AUGUUUACCAUUUaaUCTGCAGaaaaaaaaaaaaaaaaaaaaa mXR7, mXR8 and mXR8_FM mRNAs (Antoun et al, 2006b) with the upstream sequence similar to that of 002 mRNA (La Teana et al, 1993) had a strong UAAGGAGGU SD sequence marked in bold. mSD022 mRNA with the upstream sequence identical to that of 022 mRNA (La Teana et al, 1993) had a weak AGGU SD sequence (in bold). mBar mRNA with the upstream sequence identical to that of barI mRNA (Ontiveros et al, 1997) had a scrambled SD sequence AACGAG (in bold). Phenylalanine codons are marked as underlined bold while the initiation codons (canonical and non-canonical) as underlined italic. All mRNAs except mXR8_FM had MFTI coding sequence. mXR8_FM mRNA had the MTI coding sequence. Addition of both IF3 and IF1 to vacant ribosomes, or to 70S ribosomes pre-incubated with either mXR7 or mBar (Figure 1B) resulted in an about 25-fold faster splitting, than when only IF3 was added to the same ribosomal complexes (Figure 1A and Table I). We note that the IF1/IF3-dependent splitting of ribosomes pre-incubated with mXR7 was remarkably fast (0.92 s−1) and that splitting of vacant ribosomes by the simultaneous addition of IF1, IF3 and mXR7 was significantly slower (0.53 s−1) but still sufficiently fast to be of physiological relevance (Figure 1B and Table I). Pre-incubation of 70S ribosomes with deacylated tRNAPhe slightly reduced the rate of their splitting by IF1/IF3, whereas the pre-incubation with both mXR7 and tRNAPhe reduced the splitting rate to virtually zero (Figure 1B and Table I). Simultaneous addition of RRF, EF-G and IF3 to vacant ribosomes increased their splitting rate about 30-fold over that in the absence of RRF and EF-G (Table I). The splitting rates of 70S ribosomes pre-incubated with only tRNAPhe, only mXR7 or both tRNAPhe and mXR7 were significantly larger, moderately smaller or greatly larger, respectively, than when RRF, EF-G and IF3 were added to vacant ribosomes (Figure 1C and Table I). Comparison of Figure 1B and C reveals that the splitting of ribosome complexes by RRF/EF-G on the one hand and by IF1/IF3 on the other were complementary. In other words, the most rapid splitting by RRF/EF-G occurred for ribosomes in complex with both mXR7 and tRNAPhe (Figure 1C), where the splitting by IF1/IF3 was negligible (Figure 1B) and the most rapid splitting by IF1/IF3 occurred for ribosomes in complex with only mXR7 (Figure 1B), where the splitting by RRF/EF-G was the slowest (Figure 1C). Concentration dependencies of IF1/IF3- and RRF/EF-G-induced ribosome splitting The total concentrations of IF1, RRF and EF-G in the E. coli cytoplasm have been estimated to be about 10 μM (Howe and Hershey, 1983), 20 and 20 μM, respectively, (Andersen et al, 1999; Seo et al, 2004), but their free concentrations are unknown. To study how the rate of ribosome splitting by IF1/IF3 or by RRF/EF-G depends on the concentration of these factors at a fixed concentration of IF3, we performed IF1 titrations, RRF titrations at a fixed EF-G concentration and EF-G titrations at a fixed RRF concentration. Figure 2A shows how the apparent splitting rate, Q, and the extent of splitting of vacant ribosomes by IF1/IF3 increased with increasing IF1 concentration. The actual rate constant, q, for ribosome splitting, was calculated according to the equation: Figure 2.Effect of IF1 concentration on the rate of the IF1/IF3-induced splitting of vacant ribosomes and 70S·mRNA complexes. (A) Vacant 70S ribosomes (0.3 μM) or (B) 70S ribosomes (0.3 μM) pre-incubated with 0.5 μM mXR7 mRNA were rapidly mixed with the mixture containing 2 μM IF3 and different concentrations of IF1. All mixtures were prepared in LS4 buffer. (C) The splitting rate q was plotted versus IF1 concentration and fitted to the Michaelis–Menten equation: q=qcat[IF1]/([IF1+KM]). Download figure Download PowerPoint where feq is the fraction of non-dissociated 70S ribosomes in equilibrium (see Materials and methods). The increase in q was hyperbolic with a KM value (KM(IF1)) of 22 μM and a maximal rate qcat (qcat(IF1)) of 0.34 s−1 (Figure 2C and Table III). Table 3. qcat and KM parameters for the splitting of ribosomal complexes by IF1/IF3 and RRF/EF-G Splitting with Titration with Complex qcat (s−1) KM (μM) IF1/IF3 IF1 (IF3=2 μM) Vacant ribosome 0.34±0.05 22±4 IF1/IF3 IF1 (IF3=2 μM) 70S:mXR7 1.3±0.2 1.8±0.4 RRF/EF-G RRF (EF-G=3 μM; IF3=1 μM) Vacant ribosome 0.18±0.03 3.2±0.4 RRF/EF-G EF-G (RRF=5 μM; IF3=1 μM) Vacant ribosome 0.20±0.04 2.8±0.7 RRF/EF-G RRF (EF-G=3 μM; IF3=2 μM) 70S:mXR7:tRNAPhe 1.7±0.2 9.8±1.3 RRF/EF-G EF-G (RRF=5 μM; IF3=2 μM) 70S:mXR7:tRNAPhe 0.8±0.2 0.9±0.2 All experiments were conducted at 37°C in LS4 buffer containing 4 mM Mg2+. When the 70S ribosomes were pre-incubated with mXR7 mRNA with a strong SD sequence before addition of IF3 at a fixed and IF1 at a varying concentration (Figure 2B), the maximal rate qcat(IF1)=1.3 s−1 of ribosome splitting was much larger and KM(IF1)=1.8 μM much smaller than for vacant ribosomes (Figure 2C and Table III). The rate constant, q, for vacant ribosome splitting by EF-G at a fixed (3 μM) and RRF at a varying concentration in the presence of IF3 (1 μM), calculated from the apparent rate, Q, of the ribosomal splitting in Figure 3A, increased hyperbolically with the RRF concentration with a qcat value (qcat(RRF)) of 0.18 s−1 and a KM value (KM(RRF)) of 3.2 μM (Figure 3C and Table III). When the same type of titration experiment with EF-G fixed at 3 μM and RRF at a varying concentration was conducted with the ribosomes pre-incubated with mXR7 and tRNAPhe (Figure 3B), we obtained qcat(RRF)=1.7 s−1 and KM(RRF)=9.8 μM (Figure 3C and Table III). Figure 3.Effects of RRF and EF-G concentrations on the rate of RRF/EF-G-dependent splitting of vacant ribosomes and 70S·mXR7·tRNAPhe complexes. (A, D) Vacant 70S ribosomes (0.2 μM) or (B, E) 0.2 μM 70S ribosomes pre-incubated with 0.4 μM mXR7 mRNA and 0.4 μM tRNAPhe were rapidly mixed with the mixture containing different concentrations of RRF, 3 μM EF-G, 1 μM IF3 (A) or 2 μM IF3 (B) or with the mixture containing different concentrations of EF-G, 5 μM RRF, 1 μM IF3 (D) or 2 μM IF3 (E). All mixtures were prepared in LS4 buffer. (C, F) The splitting rate q plotted versus the concentration, [S], of RRF (C) or EF-G (F) was fitted to the Michaelis–Menten equation q=qcat[S]/([S]+KM(S)) Download figure Download PowerPoint When, instead, vacant ribosome splitting was studied with RRF at a fixed (5 μM) and EF-G at a varying concentration (Figure 3D), a KM value (KM(EF−G)) of 2.8 μM and a kcat value (qcat(EF−G)) of 0.2 s−1 for the splitting reaction were obtained (Figure 3F). After pre-incubation of ribosomes with mXR7 mRNA and deacylated tRNAPhe, we obtained qcat(EF-G)=0.8 s−1 and the KM(EF-G)=0.9 μM from a similar titration experiment (Figure 3E and F). The different values of qcat(RRF)=1.7 s−1 and qcat(EF-G)=0.8 s−1 obtained for the 70S·mXR7·RNAPhe complex are explained by the near-saturating EF-G concentration in the RRF titration experiment and the sub-saturating RRF concentration in the EF-G titration experiment. Role of IF3 in IF1/IF3-induced ribosome splitting Dissociation of 70S ribosomes into 30S and 50S subunits can be described by the scheme: Here, q and k are compounded rate constants for the dissociation (q) and association (k) of the 30S and 50S subunits, which depend on the presence and concentrations of IFs, mRNA and other factors (Grunberg-Manago et al, 1975). Subunit association experiments shown in Figure 4A demonstrate that the addition of IF1 (up to 15 μM) resulted in a small increase (from 3.6 to 5.4 μM−1 s−1) in k for the association of vacant subunits and in a slight decrease (from 2.1 to 1.8 μM−1 s−1) in k for the association of 30S·mRNA complexes to 50S subunits. Earlier experiments with vacant ribosomes demonstrated that the addition of IF3 greatly decreased k but left q virtually unaltered (Grunberg-Manago et al, 1975). If one assumes that IF3 does not affect q also in the presence of IF1 one can estimate the expected equilibrium dissociation constant Kd=q/k in the absence of IF3 for different IF1 concentrations using the values of k measured in experiments in Figure 4A and values of q measured in experiments in Figure 2. Taking values k∼1.8 μM−1 s−1 and q∼1 s−1 measured at 15 μM IF1 one gets Kd∼0.6 μM, which would imply that a considerable dissociation of 70S·mRNA complexes should occur at this IF1 concentration even in the absence of IF3. Figure 4.Effects of IF1 on the association/dissociation kinetics of vacant ribosomes or 70S·mRNA complexes. (A) 30S subunits (0.2 μM) (closed symbols) or 30S·mXR7 complexes (open symbols) containing no IF1 (⧫,◊), 1 μM IF1 (▪,□) or 15 μM IF1 (•,○) were mixed with 0.2 μM 50S subunits. (B) 30S·mRNA complexes (0.2 μM) containing 5 μM IF1 (Δ) or 15 μM IF1 (○) were mixed with 0.2 μM 50S subunits; 0.2 μM 70S ribosomes were mixed with 20 μM IF1 (▪); 0.2 μM 70S·mXR7 complexes were mixed with 5 μM IF1 (▴) or 15 μM IF1 (•) or with 15 μM IF1 and 2 μM IF3 (⧫). All mixtures were prepared in LS3 buffer containing 3 mM free Mg2+. Download figure Download PowerPoint Figure 4B shows, in contrast, that without IF3 the extent of dissociation of 0.2 μM 70S·mXR7 complex by 15 μM IF1 was not the 80% expected from the Kd value of 0.6 μM but only 20%, which corresponds to the Kd value of about 0.01 μM (Equation (7) in Materials and methods). This 60-fold discrepancy between the expected and measured Kd values indicates that the rate constant q of 70S·mXR7 splitting by IF1 was greatly affected by IF3, which implies a direct interaction of IF3 with the 70S·mRNA IF1 complex. This conclusion is further validated by the comparison of the q value of 1 s−1 measured in the presence of both IF1 and IF3 (Figure 4B) with the q value of 0.018 s−1 calculated according to Equation (1) from the feq value of 0.8 and the apparent rate Q of 0.16 s−1 measured for 70S·mRNA splitting by IF1 alone (Figure 4B). The extent of vacant ribosome dissociation by 20 μM IF1 was small (Figure 4B), making the precision of the Kd and q estimates low. We could, however, estimate the upper bound for q (<0.006 s−1) in the absence of IF3 from the fraction (feq∼0.93) of remaining 70S ribosomes and the apparent splitting rate Q (∼0.14 s−1). In the presence of IF3, in contrast, we estimated q as 0.15 s−1 (Figure 2A). This implies an at least 20-fold increase in the rate q of IF1-dependent splitting of vacant ribosomes by IF3, which shows that IF3 interacts directly not only with the 70S·mRNA·IF1 but also with the 70S·IF1 complex and promotes their splitting into subunits (see Discussion). Roles of IF3 and deacylated tRNA in ribosome splitting by RRF and EF-G Previous results (Karimi et al, 1999; Peske et al, 2005) demonstrated that IF3 is not directly involved in the mechanism of RRF/EF-G-dependent ribosome splitting. Indeed, splitting of both vacant and post-termination-like (PTL) ribosomes by RRF and EF-G occurred in the absence and presence of IF3 (Figure 5A and Table IV). Although the apparent rate, Q, for vacant ribosome splitting decreased from 0.95 to 0.22 s−1 by inclusion of IF3 in the assay, the actual splitting rate q, as calculated from Equation 1 above, was similar in the presence (0.13 s−1) and absence (0.16 s−1) of IF3. The apparent rate, Q, for splitting of the PTL ribosomes was 2.65 s−1 in the absence and 1.65 s−1 in the presence of IF3, whereas the corresponding q values were 1.6 and 1.5 s−1, respectively (Table IV). Moreover, the q value of about 1.5 s−1 for the PTL ribosomes remained constant, when the IF3 concentration was increased up to 2 μM, and then decreased slightly upon further increase in the IF3 concentration (results not shown). The unchanging q values upon IF3 addition confirm that IF3 did not participate in RRF/EF-G-catalysed ribosome splitting into subunits, but merely blocked subunit re-association (Karimi et al, 1999; Peske et al, 2005). Figure 5.Effects of IF3, Mg2+, mRNA and tRNA identity on the ribosomal splitting by RRF/EF-G and IF1/IF3. (A) Mixture 1 containing 0.2 μM vacant 70S ribosomes (▵,▴) or 0.2 μM 70S ribosomes pre-incubated with 0.5 μM mXR7 mRNA and 0.4 μM deacylated tRNAPhe (○,•,▪) was rapidly mixed with mixture 2 containing 9 μM RRF, 4 μM EF-G and no IF3 (open symbols) or 0.3 μM IF3 (closed symbols). (B) Mixture 1 containing 0.2 μM 70S ribosomes pre-incubated with 0.5 μM of mXR7 (•,○) or mBar (♦,⋄) mRNAs together with 0.4 μM tRNAPhe (closed symbols) or 0.4 μM tRNAfMet (open symbols) was rapidly mixed with mixture 2 containing 9 μM RRF, 4 μM EF-G and 0.3 μM IF3 or with mixture 2 containing 15 μM IF1 and 2 μM IF3. (C) Mixture 1 containing 0.2 μM vacant 70S ribosomes (▴), 0.2 μM 70S ribosomes pre-incubated with 0.5 μM of mSD022 (□), mBar (⧫), mXR8_FM (▪), mXR8 (◊) or mXR7 (•) mRNAs was rapidly mixed with mixture 2 containing 5 μM IF1 and 2 μM IF3. All mixtures were prepared in LS3 buffer, except that experiment marked (▪) in (A) was conducted in LS5 buffer containing 5 mM free Mg2+. Download figure Download PowerPoint Table 4. Effect of IF3 on the RRF/EF-G-dependent splitting of vacant ribosomes and 70S·mXR7·tRNAPhe complexes feq Q (s−1) q (s−1) Vacant 70S ribosome (−IF3) 0.7 0.95±0.08 0.16±0.04 Vacant 70S ribosome (+IF3) 0.25 0.22±0.02 0.13±0.01 70S·mXR7·tRNAPhe (−IF3) 0.24 2.65±0.08 1.60±0.05 70S·mXR7·tRNAPhe (+IF3) 0.05 1.65±0.07 1.51±0.06 Apparent rate of ribosome splitting, Q, was obtained from nonlinear regression fit of experimental data to the relation (Equation (8) in Materials and methods) after which q was calculated from Equation (1). All experiments were conducted at 37°C in LS3 buffer containing 3 mM Mg2+. To accurately monitor the time course of ribosome splitting in the absence of IF3, the concentration of free Mg2+ in the experiments in Figure 5 was reduced from 4 to 3 mM, which also led to faster ribosome splitting (compare Tables I and IV). This effect of Mg2+ was corroborated by the experiment in Figure 5A showing that the increase in Mg2+ concentration from 3 to 5 mM under otherwise identical conditions decreased the splitting rate q for the 70S·mXR7·tRNAPhe complex from 1.5 to 0.7 s−1. The rate of splitting of PTL ribosomes depended significantly on the identity of the deacylated tRNA. PTL ribosomes with different mRNAs (Table II) containing tRNAfMet in the P site were split at least three-fold more slowly than those containing tRNAPhe (Table V), as illustrated for the mBar and mXR7 cases in Figure 5B. The mRNAs in these PTL ribosomes had different distances between their SD sequences and the Met or Phe codons (mXR8, mXR8_FM) and varying strength of the SD sequences (mXR7, mBar, mSD022) (Table II), which excludes the possibility that peculiarities of an mRNA could account for the slower splitting of the tRNAfMet-containing PTL ribosomes. Stoichiometric formation of PTL ribosomes was confirmed for the different types of mRNA and tRNA in control experiments verifying lack of splitting by IF1/IF3 (Figure 5B), typical for the PTL ribosomes (Figure 1B). Table 5. Effects of mRNA and tRNA identity on the splitting of ribosomal complexes by RRF/EF-G and IF1/IF3 Splitting by RRF/EF-G A1 (%) q1 (s−1) A2 (%) q2 (s−1) 70S:mRNA:tRNAPhe with mXR7 85 1.77±0.06 15 0.33±0.03 mXR8 81 1.72±0.06 19 0.32±0.03 mXR8_FM 78 1.44±0.05 22 0.33±0.04 mBar 55 2.8±0.08 45 0.31±0.05 mSD022 65 1.47±0.06 35 0.29±0.04 70S:mRNA:tRNAfMet with mXR7 88 0.51±0.02 12 0.12±0.03 mXR8 85 0.48±0.02 15 0.13±0.02 mXR8_FM 47 0.47±0.05 53 0.18±0.03 mBar 73 0.48±0.03 27 0.11±0.02 mSD022 71 0.51±0.03 29 0.15±0.03 70S:mRNA:tRNAfMet prepared from initiation complexes with mXR7 92 0.54±0.02 8 0.09±0.02 mXR8 86 0.51±0.02 14 0.08±0.02 mXR8_FM 55 0.41±0.04 45 0.08±0.03 mBar 64 0.50±0.04 36 0.11±0.03 mSD022 81 0.48±0.03 19 0.09±0.02 Splitting by IF1/IF3 A1 q1 (s−1) A2 q2 (s−1) 70S 81 0.07±0.03 19 0.03±0.005 70S:mXR7 87 1.23±0.06 13 0.08±0.02 70S:mXR8 83 0.81±0.05 17 0.07±0.01 70S:mXR8_FM 76 1.05±0.06 24 0.06±0.01 70S:mBar 59 0.28±0.04 41 0.05±0.02 70S:mSD022 80 0.11±0.03 20 0.04±0.005 Parameters A1, A2, q1 and q2 were determined from nonlinear regression fit of experimental data to the relation $I(t) = A_{1} exp ( - q_{1} t) + A_{2} exp ( - q_{2} t) + A_{{{rm BKG}}} $ (Equation (10) in Materials and methods). All experiments were conducted at 37°C in LS3 buffer containing 3 mM Mg2+. A near-complete splitting of PTL ribosomes by RRF/EF-G in the presence of IF3 (feq<0.08) justifies the use of the sum of exponentials to fit light-scattering data (see Materials and methods for details). Figure 5B shows that RRF/EF-G splitting of 70S·mRNA·tRNA complexes with mBar mRNA was distinctly bi-phasic, whereas splitting of complexes with mXR7 mRNA was mainly mono-phasic for both tRNAfMet and tRNAPhe (Table V). Distinct bi-phasic splitting by RRF/EF-G was also observed for some complexes formed with mXR8_FM and mSD022 mRNAs. Qualitatively similar bi-phasic behaviour has previously been observed in fast kinetics studies of EF-G-dependent translocation with pre-translocation ribosomes assembled on mSD022-like mRNAs (Peske et al, 2004). It was suggested that the slow phase is related to ribosomes with the 30S subunit in inactive conformation and that the fast phase is related to EF-G-dependent translocation on ribosomes with the 30S subunit in active conformation (Peske et al, 2004). Accordingly, we suggest that the fast phase is related to RRF/EF-G-dependent splitting of fully active PTL ribosomes. In line with this, we found similar fast phase rate and amplitude for the RRF/G-dependent splitting (Table V) when the PTL ribosomes were prepared by puromycin treatment of 70S·mRNA·fMet-tRNAfMet initiation complexes f