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

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.     

Localization of the trigger factor binding site on the ribosomal 50S subunit

In Escherichia coli, protein folding is undertaken by three distinct sets of chaperones, the DnaK-DnaJ and GroEL-GroES systems and the trigger factor (TF). TF has been proposed to be the first chaperone to interact with the nascent polypeptide chain as it emerges from the tunnel of the 70S ribosome and thus probably plays an important role in co-translational protein folding. We have made complexes with deuterated ribosomes (50S subunits and 70S ribosomes) and protated TF and determined the TF binding site on the respective complexes using the neutron scattering technique of spin-contrast variation. Our data suggest that the TF binds in the form of a homodimer. On both the 50S subunit and the 70S ribosome, the TF position is in proximity to the tunnel exit site, near ribosomal proteins L23 and L29, located on the back of the 50S subunit. The positions deviate from one another, such that the position on the 70S ribosome is located slightly further from the tunnel than that determined for the 50S subunit alone. Nevertheless, from both determined positions interaction between TF and a short nascent chain of 57 amino acid residues would be plausible, compatible with a role for TF participation in co-translational protein folding.     

Structural Insights into ribosome recycling factor interactions with the 70S ribosome

At the end of translation in bacteria, ribosome recycling factor (RRF) is used together with elongation factor G to recycle the 30S and 50S ribosomal subunits for the next round of translation. In x-ray crystal structures of RRF with the Escherichia coli 70S ribosome, RRF binds to the large ribosomal subunit in the cleft that contains the peptidyl transferase center. Upon binding of either E. coli or Thermus thermophilus RRF to the E. coli ribosome, the tip of ribosomal RNA helix 69 in the large subunit moves away from the small subunit toward RRF by 8 Å, thereby disrupting a key contact between the small and large ribosomal subunits termed bridge B2a. In the ribosome crystals, the ability of RRF to destabilize bridge B2a is influenced by crystal packing forces. Movement of helix 69 involves an ordered-to-disordered transition upon binding of RRF to the ribosome. The disruption of bridge B2a upon RRF binding to the ribosome seen in the present structures reveals one of the key roles that RRF plays in ribosome recycling, the dissociation of 70S ribosomes into subunits. The structures also reveal contacts between domain II of RRF and protein S12 in the 30S subunit that may also play a role in ribosome recycling.     

Rapid kinetic analysis of EF-G-dependent mRNA translocation in the ribosome

Precise and coordinated movement of the tRNA-mRNA complex within the ribosome is a fundamental step during protein biosynthesis. The molecular mechanism for this process is still poorly understood. Here we describe a new sensitive method for monitoring elongation factor G-dependent translocation of the mRNA in the ribosome. In this method, the fluorescent probe pyrene is covalently attached to the 3' end of a short mRNA sequence at position +9. Translocation of the mRNA by one codon results in a significant decrease in the fluorescence emission of pyrene and can be used to directly monitor mRNA movement using rapid kinetic methods. Importantly, this method offers the flexibility of using any tRNA or tRNA analog in order to elucidate the molecular mechanism of translocation. Our results show that the mRNA is translocated at the same rate as the tRNAs, which is consistent with the view that the movement of the tRNAs and the mRNA are coupled in the ribosome. Furthermore, an anticodon stem-loop analog of tRNA is translocated from the ribosomal A site at a rate constant that is 350-fold lower than peptidyl tRNA, indicating that the D stem, T stem and acceptor stem of A site tRNA contribute significantly to the rate of translocation.     

Specific binding of ribosome recycling factor (RRF) with the Escherichia coli ribosomes by BIACORE

The direct assays on Biacore with immobilised RRF and purified L11 from E. coli in the flow trough have shown unspecific binding between the both proteins. The interaction of RRF with GTPase domain of E. coli ribosomes, a functionally active complex of L11 with 23S r RNA and L10.(L7/L12)₄ was studied by Biacore. In the experiments of binding of RRF with 30S, 50S and 70S ribosomes from E. coli were used the antibiotics thiostrepton, tetracycline and neomycin and factors, influencing the 70S dissociation Mg²⁺, NH₄Cl, EDTA. The binding is strongly dependent from the concentrations of RRF, Mg²⁺, NH₄Cl, EDTA and is inhibited by thiostrepton. The effect is most specific for 50S subunits and indicates that the GTPase centre can be considered as a possible site of interaction of RRF with the ribosome. We can consider an electrostatic character of the interactions with most probable candidate 16S and 23S r RNA at the interface of 30S and 50S ribosomal subunits.     

Enzymatic binding of aminoacyl-tRNA and peptide chain elongation by ribosomes and ribosome subunits in pea

In vitro aminoacyl transfer from aminoacyl-tRNA to elongating peptide chains and binding of aminoacyl-tRNA to ribosomes were studied with n     

Functional interactions by transfer RNAs in the ribosome

Recent X-ray crystal structures of the ribosome have revolutionized the field by providing a much-needed structural framework to understand ribosome function. Indeed, the crystal structures rationalize much of the genetic and biochemical data that have been meticulously gathered over 50 years. Here, we focus on the interactions between tRNAs and the ribosome and describe some of the insights that the structures provide about the mechanism of translation. Both high-resolution structures and functional studies are essential for fully appreciating the complex process of protein synthesis.     

Combining electron crystallography and X-ray crystallography to study the MlotiK1 cyclic nucleotide-regulated potassium channel

We have recently reported the X-ray structure of the cyclic nucleotide-regulated potassium channel, MlotiK1. Here we describe the application of both electron and X-ray crystallography to obtain high quality crystals. We suggest that the combined application of these techniques provides a useful strategy for membrane protein structure determination. We also present negative stain projection and cryo-data projection maps. These maps provide new insights about the properties of the MlotiK1 channel. In particular, a comparison of a 9 Å cryo-data projection with calculated model maps strongly suggests that there is a very weak interaction between the pore and the S1–S4 domains of this 6 TM tetrameric cation channel and that the S1–S4 domains can adopt multiple orientations relative to the pore.     

Site-specific cleavage of the 40S ribosomal subunit reveals eukaryote-specific ribosomal protein S28 in the subunit head

After resolving the crystal structure of the prokaryotic ribosome, mapping the proteins in the eukaryotic ribosome is a challenging task. We applied RNase H digestion to split the human 40S ribosomal subunit into head and body parts. Mass spectrometry of the proteins in the 40S subunit head revealed the presence of eukaryote-specific ribosomal protein S28e. Recombinant S28e was capable of specific binding to the 3′ major domain of the 18S rRNA (K_a = 8.0 ± 0.5 × 10⁹ M⁻¹). We conclude that S28e has a binding site on the 18S rRNA within the 40S subunit head.

Structured summary

MINT-8044084: S8 (uniprotkb:P62241) and S19 (uniprotkb:P39019) colocalize (MI:0403) by cosedimentation through density gradient (MI:0029)MINT-8044095: S8 (uniprotkb:P62241), S19 (uniprotkb:P39019) and S13 (uniprotkb:P62277) colocalize (MI:0403) by cosedimentation through density gradient (MI:0029)MINT-8044024: S29 (uniprotkb:P62273), S28 (uniprotkb:P62857), S21 (uniprotkb:P63220), S20 (uniprotkb:P60866), S26 (uniprotkb:P62854), S25 (uniprotkb:P62851), S12 (uniprotkb:P25398), S17 (uniprotkb:P08708), S19 (uniprotkb:P39019), S14 (uniprotkb:P62263), S16 (uniprotkb:P62249) and S11 (uniprotkb:P62280) colocalize (MI:0403) by cosedimentation through density gradient (MI:0029)MINT-8044065: S29 (uniprotkb:P62273), S28 (uniprotkb:P62857), S19 (uniprotkb:P39019), S14 (uniprotkb:P62263) and S16 (uniprotkb:P62249) colocalize (MI:0403) by cosedimentation through density gradient (MI:0029)     

Conformational changes in switch I of EF‐G drive its directional cycling on and off the ribosome

We have trapped elongation factor G (EF-G) from Escherichia coli in six, functionally defined states, representing intermediates in its unidirectional catalytic cycle, which couples GTP hydrolysis to tRNA–mRNA translocation in the ribosome. By probing EF-G with trypsin in each state, we identified a substantial conformational change involving its conserved switch I (sw1) element, which contacts the GTP substrate. By attaching FeBABE (a hydroxyl radical generating probe) to sw1, we could monitor sw1 movement (by ∼20 Å), relative to the 70S ribosome, during the EF-G cycle. In free EF-G, sw1 is disordered, particularly in GDP-bound and nucleotide-free states. On EF-G•GTP binding to the ribosome, sw1 becomes structured and tucked inside the ribosome, thereby locking GTP onto EF-G. After hydrolysis and translocation, sw1 flips out from the ribosome, greatly accelerating release of GDP and EF-G from the ribosome. Collectively, our results support a central role of sw1 in driving the EF-G cycle during protein synthesis.     

Structure and function of FusB: an elongation factor G-binding fusidic acid resistance protein active in ribosomal translocation and recycling

Fusidic acid (FA) is a bacteriostatic antibiotic that locks elongation factor G (EF-G) to the ribosome after GTP hydrolysis during elongation and ribosome recycling. The plasmid pUB101-encoded protein FusB causes FA resistance in clinical isolates of Staphylococcus aureus through an interaction with EF-G. Here, we report 1.6 and 2.3 Å crystal structures of FusB. We show that FusB is a two-domain protein lacking homology to known structures, where the N-terminal domain is a four-helix bundle and the C-terminal domain has an alpha/beta fold containing a C4 treble clef zinc finger motif and two loop regions with conserved basic residues. Using hybrid constructs between S. aureus EF-G that binds to FusB and Escherichia coli EF-G that does not, we show that the sequence determinants for FusB recognition reside in domain IV and involve the C-terminal helix of S. aureus EF-G. Further, using kinetic assays in a reconstituted translation system, we demonstrate that FusB can rescue FA inhibition of tRNA translocation as well as ribosome recycling. We propose that FusB rescues S. aureus from FA inhibition by preventing formation or facilitating dissociation of the FA-locked EF-G–ribosome complex.     

Visualization of tRNA movements on the Escherichia coli 70S ribosome during the elongation cycle

Three-dimensional cryomaps have been reconstructed for tRNA-ribosome complexes in pre- and posttranslocational states at 17-A resolution. The positions of tRNAs in the A and P sites in the pretranslocational complexes and in the P and E sites in the posttranslocational complexes have been determined. Of these, the P-site tRNA position is the same as seen earlier in the initiation-like fMet-tRNA(f)(Met)-ribosome complex, where it was visualized with high accuracy. Now, the positions of the A- and E-site tRNAs are determined with similar accuracy. The positions of the CCA end of the tRNAs at the A site are different before and after peptide bond formation. The relative positions of anticodons of P- and E-site tRNAs in the posttranslocational state are such that a codon-anticodon interaction at the E site appears feasible.     

The C-terminal helix in the YjeQ zinc-finger domain catalyzes the release of RbfA during 30S ribosome subunit assembly

YjeQ (also called RsgA) and RbfA proteins in Escherichia coli bind to immature 30S ribosome subunits at late stages of assembly to assist folding of the decoding center. A key step for the subunit to enter the pool of actively translating ribosomes is the release of these factors. YjeQ promotes dissociation of RbfA during the final stages of maturation; however, the mechanism implementing this functional interplay has not been elucidated. YjeQ features an amino-terminal oligonucleotide/oligosaccharide binding domain, a central GTPase module and a carboxy-terminal zinc-finger domain. We found that the zinc-finger domain is comprised of two functional motifs: the region coordinating the zinc ion and a carboxy-terminal α-helix. The first motif is essential for the anchoring of YjeQ to the 30S subunit and the carboxy-terminal α-helix facilitates the removal of RbfA once the 30S subunit reaches the mature state. Furthermore, the ability of the mature 30S subunit to stimulate YjeQ GTPase activity also depends on the carboxy-terminal α-helix. Our data are consistent with a model in which YjeQ uses this carboxy-terminal α-helix as a sensor to gauge the conformation of helix 44, an essential motif of the decoding center. According to this model, the mature conformation of helix 44 is sensed by the carboxy-terminal α-helix, which in turn stimulates the YjeQ GTPase activity. Hydrolysis of GTP is believed to assist the release of YjeQ from the mature 30S subunit through a still uncharacterized mechanism. These results identify the structural determinants in YjeQ that implement the functional interplay with RbfA.     

Observation of intersubunit movement of the ribosome in solution using FRET

Protein synthesis is believed to be a dynamic process, involving structural rearrangements of the ribosome. Cryo-EM reconstructions of certain elongation factor G (EF-G)-containing complexes have led to the proposal that translocation of tRNA and mRNA through the ribosome, from the A to P to E sites, is accompanied by a rotational movement between the two ribosomal subunits. Here, we have used F?rster resonance energy transfer (FRET) to monitor changes in the relative orientation of the ribosomal subunits in different complexes trapped at intermediate stages of translocation in solution. Binding of EF-G to the ribosome in the presence of the non-hydrolyzable GTP analogue GDPNP or GTP plus fusidic acid causes an increase in the efficiency of energy transfer between fluorophores introduced into proteins S11 in the 30 S subunit and L9 in the 50 S subunit, and a decrease in energy transfer between S6 and L9. Similar anti-correlated changes in energy transfer occur upon binding the GTP-requiring release factor RF3. These changes are consistent with the counter-clockwise rotation of the 30 S subunit relative to the 50 S subunit observed in cryo-EM studies. Reaction of ribosomal complexes containing the peptidyl-tRNA analogues N-Ac-Phe-tRNAPhe, N-Ac-Met-tRNAMet or f-Met-tRNAfMet with puromycin, conditions favoring movement of the resulting deacylated tRNAs into the P/E hybrid state, leads to similar changes in FRET. Conversely, treatment of a ribosomal complex containing deacylated and peptidyl-tRNAs bound in the A/P and P/E states, respectively, with EF-G.GTP causes reversal of the FRET changes. The use of FRET has enabled direct observation of intersubunit movement in solution, provides independent evidence that formation of the hybrid state is coupled to rotation of the 30 S subunit and shows that the intersubunit movement is reversed during the second step of translocation.     

Assembly of the central domain of the 30S ribosomal subunit: roles for the primary binding ribosomal proteins S15 and S8

Assembly of the 30S ribosomal subunit occurs in a highly ordered and sequential manner. The ordered addition of ribosomal proteins to the growing ribonucleoprotein particle is initiated by the association of primary binding proteins. These proteins bind specifically and independently to 16S ribosomal RNA (rRNA). Two primary binding proteins, S8 and S15, interact exclusively with the central domain of 16S rRNA. Binding of S15 to the central domain results in a conformational change in the RNA and is followed by the ordered assembly of the S6/S18 dimer, S11 and finally S21 to form the platform of the 30S subunit. In contrast, S8 is not part of this major platform assembly branch. Of the remaining central domain binding proteins, only S21 association is slightly dependent on S8. Thus, although S8 is a primary binding protein that extensively contacts the central domain, its role in assembly of this domain remains unclear. Here, we used directed hydroxyl radical probing from four unique positions on S15 to assess organization of the central domain of 16S rRNA as a consequence of S8 association. Hydroxyl radical probing of Fe(II)-S15/16S rRNA and Fe(II)-S15/S8/16S rRNA ribonucleoprotein particles reveal changes in the 16S rRNA environment of S15 upon addition of S8. These changes occur predominantly in helices 24 and 26 near previously identified S8 binding sites. These S8-dependent conformational changes are consistent with 16S rRNA folding in complete 30S subunits. Thus, while S8 binding is not absolutely required for assembly of the platform, it appears to affect significantly the 16S rRNA environment of S15 by influencing central domain organization.     

The sarcin-ricin loop of 23S rRNA is essential for assembly of the functional core of the 50S ribosomal subunit

The sarcin–ricin loop (SRL) of 23S rRNA in the large ribosomal subunit is a factor-binding site that is essential for GTP-catalyzed steps in translation, but its precise functional role is thus far unknown. Here, we replaced the 15-nucleotide SRL with a GAAA tetraloop and affinity purified the mutant 50S subunits for functional and structural analysis in vitro. The SRL deletion caused defects in elongation-factor-dependent steps of translation and, unexpectedly, loss of EF-Tu-independent A-site tRNA binding. Detailed chemical probing analysis showed disruption of a network of rRNA tertiary interactions that hold together the 23S rRNA elements of the functional core of the 50S subunit, accompanied by loss of ribosomal protein L16. Our results reveal an influence of the SRL on the higher-order structure of the 50S subunit, with implications for its role in translation.