Structural study of translating 70 S ribosomes from Escherichia coli: I. Electron microscopy

Translating 70 S ribosomes of Escherichia coli either in the pre-translocation or in the post-translocation state have been prepared by using the cell-free translation system in poly(U)—S—S—Sepharose columns [Methods Enzymol. (1979) 59, 382–398]. Electron microscopy study of the preparations has demonstrated that: (1) the mutual orientation of the ribosomal subunits in the translating ribosomes is the same as proposed by Lake for routine 30 S·50 S couples [J. Mol. Biol. (1976) 105, 111–130]; (2) the L7/L12 stalk of the 50 S subunit sticks out from the 70 S particle and does not join the 30 S subunit; (3) pre-translocation and post-translocation state ribosomes do not differ in mutual orientation of the subunits and in the position of the L7/L12 stalk, within the limits of electron microscopy resolution.     

Structural studies of translating ribosomes. II. Comparative sedimentation analysis of pre- and post-translocation states

Using the solid-phase translation system technique where template poly(U) is covalently coupled to Sepharose through cleavable disulfide bridges translating monoribosomes carrying a polypeptide (polyPhe) of 10 to 20 amino acids long have been isolated. Both pre-translocation state and post-translocation state ribosomes have been obtained. It has been shown that the sedimentation coefficient of the pre-translocation state ribosomes exceeds that of the post-translocation state ribosomes by a magnitude of about 1S. This difference is independent on the sedimentation rate (hydrostatic pressure) in the range of 20 000 to 40 000 rev/min and, most likely, is not a direct contribution of the increase of the particle mass at the expense of an additional tRNA in the pre-translocation state ribosomes. Together with other data, this result suggests that translating ribosomes in the pre-translocation state are more compact than post-translocation state ribosomes.     

Direct observation of translocation in individual DNA polymerase complexes

Complexes of phi29 DNA polymerase and DNA fluctuate on the millisecond time scale between two ionic current amplitude states when captured atop the α-hemolysin nanopore in an applied field. The lower amplitude state is stabilized by complementary dNTP and thus corresponds to complexes in the post-translocation state. We have demonstrated that in the upper amplitude state, the DNA is displaced by a distance of one nucleotide from the post-translocation state. We propose that the upper amplitude state corresponds to complexes in the pre-translocation state. Force exerted on the template strand biases the complexes toward the pre-translocation state. Based on the results of voltage and dNTP titrations, we concluded through mathematical modeling that complementary dNTP binds only to the post-translocation state, and we estimated the binding affinity. The equilibrium between the two states is influenced by active site-proximal DNA sequences. Consistent with the assignment of the upper amplitude state as the pre-translocation state, a DNA substrate that favors the pre-translocation state in complexes on the nanopore is a superior substrate in bulk phase for pyrophosphorolysis. There is also a correlation between DNA sequences that bias complexes toward the pre-translocation state and the rate of exonucleolysis in bulk phase, suggesting that during DNA synthesis the pathway for transfer of the primer strand from the polymerase to exonuclease active site initiates in the pre-translocation state.     

Dynamics of Translocation and Substrate Binding in Individual Complexes Formed with Active Site Mutants of Φ29 DNA Polymerase

The Φ29 DNA polymerase (DNAP) is a processive B-family replicative DNAP. Fluctuations between the pre-translocation and post-translocation states can be quantified from ionic current traces, when individual Φ29 DNAP-DNA complexes are held atop a nanopore in an electric field. Based upon crystal structures of the Φ29 DNAP-DNA binary complex and the Φ29 DNAP-DNA-dNTP ternary complex, residues Tyr-226 and Tyr-390 in the polymerase active site were implicated in the structural basis of translocation. Here, we have examined the dynamics of translocation and substrate binding in complexes formed with the Y226F and Y390F mutants. The Y226F mutation diminished the forward and reverse rates of translocation, increased the affinity for dNTP in the post-translocation state by decreasing the dNTP dissociation rate, and increased the affinity for pyrophosphate in the pre-translocation state. The Y390F mutation significantly decreased the affinity for dNTP in the post-translocation state by decreasing the association rate ∼2-fold and increasing the dissociation rate ∼10-fold, implicating this as a mechanism by which this mutation impedes DNA synthesis. The Y390F dissociation rate increase is suppressed when complexes are examined in the presence of Mn²⁺ rather than Mg²⁺. The same effects of the Y226F or Y390F mutations were observed in the background of the D12A/D66A mutations, located in the exonuclease active site, ∼30 Å from the polymerase active site. Although translocation rates were unaffected in the D12A/D66A mutant, these exonuclease site mutations caused a decrease in the dNTP dissociation rate, suggesting that they perturb Φ29 DNAP interdomain architecture.     

A fast dynamic mode of the EF‐G‐bound ribosome

A key intermediate in translocation is an ‘unlocked state’ of the pre‐translocation ribosome in which the P‐site tRNA adopts the P/E hybrid state, the L1 stalk domain closes and ribosomal subunits adopt a ratcheted configuration. Here, through two‐ and three‐colour smFRET imaging from multiple structural perspectives, EF‐G is shown to accelerate structural and kinetic pathways in the ribosome, leading to this transition. The EF‐G‐bound ribosome remains highly dynamic in nature, wherein, the unlocked state is transiently and reversibly formed. The P/E hybrid state is energetically favoured, but exchange with the classical P/P configuration persists; the L1 stalk adopts a fast dynamic mode characterized by rapid cycles of closure and opening. These data support a model in which P/E hybrid state formation, L1 stalk closure and subunit ratcheting are loosely coupled, independent processes that must converge to achieve the unlocked state. The highly dynamic nature of these motions, and their sensitivity to conformational and compositional changes in the ribosome, suggests that regulating the formation of this intermediate may present an effective avenue for translational control.     

The mechanism of the protein-synthesis elongation cycle in eukaryotes. Effect of ricin on the ribosomal interaction with elongation factors

The functional significance of the post-translocation interaction of eukaryotic ribosomes with EF-2 was studied using the translational inhibitor ricin. Ribosomes treated with ricin showed a decreased rate of elongation accompanied by altered proportions of the different ribosomal phases of the elongation cycle. The content of ribosome-bound EF-2 was diminished by approximately 65% while that of EF-1 was unaffected. The markedly reduced content of EF-2 was caused by an inability of the ricin-treated ribosomes to form high-affinity pre-translocation complexes with EF-2. However, the ribosomes were still able to interact with EF-2 in the form of a low-affinity post-translocation complex. Ricin-treated ribosomes showed an altered ability to stimulate the GTP hydrolysis catalysed by either EF-1 or EF-2. The EF-1-catalysed hydrolysis was reduced by approximately 70%, resulting in a decreased turnover of the quaternary EF-1 X GTP X aminoacyl-tRNA X ribosome complex. In contrast, the EF-2-catalysed hydrolysis was increased by more than 400%, despite the lack of pre-translocation complex formation. The effect was not restricted to empty reconstituted ribosomes since gently salt-washed polysomes also showed an increased rate of GTP hydrolysis. The results indicate that the EF-1- and EF-2-dependent hydrolysis of GTP was activated by a common center on the ribosome that was specifically adapted for promoting the GTP hydrolysis of either EF-1 or EF-2. Furthermore, the results suggest that the GTP hydrolysis catalysed by EF-2 occurred in the low-affinity post-translocation complex.     

Neuronal RNA granules are ribosome complexes stalled at the pre-translocation state

The polarized cell morphology of neurons dictates many neuronal processes, including the axodendridic transport of specific mRNAs and subsequent translation. mRNAs together with ribosomes and RNA-binding proteins form RNA granules that are targeted to axodendrites for localized translation in neurons. It has been established that localized protein synthesis in neurons is essential for long-term memory formation, synaptic plasticity, and neurodegeneration. We have used proteomics and electron microscopy to characterize neuronal RNA granules (nRNAg) isolated from rat brain tissues or human neuroblastoma. We show that ribosome-containing RNA granules are morula-like structures when visualized by electron microscopy. Crosslinking-coupled mass-spectrometry identified a potential G3BP2 binding site on the ribosome near the eIF3d-binding site on the 40S ribosomal subunit. We used cryo-EM to resolve the structure of the ribosome-component of nRNAg. The cryo-EM reveals that predominant particles in nRNAg are 80S ribosomes, resembling the pre-translocation state where tRNA’s are in the hybrid A/P and P/E site. We also describe a new kind of principal motion of the ribosome, which we call the rocking motion.     

Ribosomal proteins S3, S6, S8 and S10 of Escherichia coli localized on the external surface of the small subunit by immune electron microscopy

The three-dimensional locations of Escherichia coli ribosomal proteins S3, 86, S8 and S10 on the surface of the small subunit were determined by immune electron microscopy.All four proteins are located on the “external surface” of the small subunit; i.e. on the side of the subunit in contact with the cytosol in the 70 S ribosome. Proteins S3, S6, S8 and S10 map at single sites, although the S3 site is extended approximately 40Å along the long axis of the subunit. S8 is located near the base of the cleft separating the platform from the upper one-third or head; protein S10 is located in the head, near the site previously mapped for S14; S3 extends from the level of the constriction to near the top of the head in the vicinity of S10; and S6 is located on the platform of the small subunit near the site previously mapped for S11.The locations of these proteins correlate well with other information on their spatial relationships obtained from assembly interactions, neutron diffraction, crosslinking and protein associations.     

Structural analysis of translating ribosomes. Proton magnetic resonance method

Comparison has been made of the proton magnetic resonance (PMR) spectra of translating ribosomes in the pre-translocation and post-translocation states as well as of the complexes of translating ribosomes with elongation factors Tu (EF-Tu) or G (EF-G) in the presence of the uncleavable analogue of GTP--guanylyl-imidodiphosphate (GMP-PNP). It is shown that proteins L7/L12 within the translating ribosomes possess a high intramolecular mobility both in the pre-translocation and in the post-translocation states. The interaction of EF-G with translating ribosomes results in a decrease of the mobility of the L7/L12 proteins. The interaction of EF-Tu with translating ribosomes leads to slight changes in the PMR spectra different from the changes caused by EF-G.     

Prokaryotic ribosomes recode the HIV-1 gag-pol-1 frameshift sequence by an E/P site post-translocation simultaneous slippage mechanism.

The mechanism favoured for -1 frameshifting at typical retroviral sites is a pre-translocation simultaneous slippage model. An alternative post-translocation mechanism would also generate the same protein sequence across the frameshift site and therefore in this study the strategic placement of a stop codon has been used to distinguish between the two mechanisms. A 26 base pair frameshift sequence from the HIV-1 gag-pol overlap has been modified to include a stop codon immediately 3' to the heptanucleotide frameshift signal, where it often occurs naturally in retroviral recoding sites. Stop codons at the 3'-end of the heptanucleotide sequence decreased the frame-shifting efficiency on prokaryote ribosomes and the recording event was further depressed when the levels of the release factors in vivo were increased. In the presence of elevated levels of a defective release factor 2, frameshifting efficiency in vivo was increased in the constructs containing the stop codons recognized specifically by that release factor. These results are consistent with the last six nucleotides of the heptanucleotide slippery sequence occupying the ribosomal E and P sites, rather than the P and A sites, with the next codon occupying the A site and therefore with a post-translocation rather than a pre-translocation -1 slippage model.     

Immune electron microscopic localization of dinitrophenyl-modified ribosomal protein S19 in reconstituted Escherichia coli 30 S subunits using antibodies to dinitrophenol

Escherichia coli small ribosomal subunits have been reconstituted from RNA and high performance liquid chromatography-purified proteins including protein S19 that had been modified at its amino-terminal proline residue with 1-fluoro-2,4-dinitrobenzene. As detailed in the accompanying paper (Olah, T. V., Olson, H. M., Glitz, D. G., and Cooperman, B. S. (1988) J. Biol. Chem. 263, 4795-4800), dinitrophenyl (DNP)-S19 was efficiently incorporated into the site ordinarily occupied by S19. Antibodies to DNP bound effectively to the reconstituted subunits and did not cause dissociation of the modified protein from the subunit. Electron microscopy of the immune complexes was used to localize the modified protein on the subunit surface. More than 95% of the antibody binding sites seen were consistent with a single location of protein S19 on the upper portion or head of the subunit, on the surface that faces the 50 S particle in a 70 S ribosome, and in an area relatively distant from the subunit platform. The S19 site is close to the region in which 30 S subunits are photoaffinity labeled with puromycin. Protein S19 is thus near protein S14 in the small subunit and in proximity to the peptidyl transferase center of the 70 S ribosome.     

Reduced puromycin sensitivity of translocated polysomes after the addition of elongation factor 2 and non-hydrolysable GTP analogues.

Treatment of reticulocyte polysomes with elongation factor eEF-2 and GTP led to an increased sensitivity of peptidyl-tRNA for puromycin as a result of the translocation from the ribosomal A-site to the P-site. Upon addition of an excess of the non-hydrolysable GTP analogue, GuoPP[CH2]P, the puromycin sensitivity decreased rapidly. The decrease in sensitivity required high concentrations of eEF-2 with half maximal effect at an eEF-2 concentration of around 1 microM. The data suggest either that peptidyl-tRNA had re-translocated back to the A-site due to the higher affinity of eEF-2 for the pre-translocation than for the post-translocation ribosome, or that the eEF-2-GuoPP[CH2]P complex blocks the peptidyl-transferase activity.     

Structural dynamics of translating ribosomes.

We describe three groups of small angle neutron scattering (SANS) experiments with translating ribosomes: 1) regular protonated (normal abundance hydrogen) particles; 2) two isotopic hybrid particles which are reconstituted from one protonated and the other deuterated subunit; 3) four isotypic hybrid particles differing from each other by the extent of protein and RNA deuteration. Using the SANS contrast variation method the radii of gyration of protein and RNA components in both ribosomal subunits as well as the intersubunit distance in the pre- and post-translocation states were determined. The results obtained suggest the following model of the ribosome as a dynamic machine. The ribosome oscillates between two major conformers differing in geometrical dimensions. The 'active' (pulsating) part of the ribosome is the 30S subunit. We believe that the movement of its 'head' relative to the passive 50S subunit is the main mechanical act of translocation. The radius of gyration of the 30S subunit and the intersubunit distance change upon the movement. This is corroborated by neutron scattering data.     

A functional interaction between ribosomal proteins S7 and S11 within the bacterial ribosome

In this study, we used site-directed mutagenesis to disrupt an interaction that had been detected between ribosomal proteins S7 and S11 in the crystal structure of the bacterial 30 S subunit. This interaction, which is located in the E site, connects the head of the 30 S subunit to the platform and is involved in the formation of the exit channel through which passes the 30 S-bound messenger RNA. Neither mutations in S7 nor mutations in S11 prevented the incorporation of the proteins into the 30 S subunits but they perturbed the function of the ribosome. In vivo assays showed that ribosomes with either mutated S7 or S11 were altered in the control of translational fidelity, having an increased capacity for frameshifting, readthrough of a nonsense codon and codon misreading. Toeprinting and filter-binding assays showed that 30 S subunits with either mutated S7 or S11 have an enhanced capacity to bind mRNA. The effects of the S7 and S11 mutations can be related to an increased flexibility of the head of the 30 S, to an opening of the mRNA exit channel and to a perturbation of the proposed allosteric coupling between the A and E sites. Altogether, our results demonstrate that S7 and S11 interact in a functional manner and support the notion that protein-protein interactions contribute to the dynamics of the ribosome.     

Structured mRNAs regulate translation initiation by binding to the platform of the ribosome

Gene expression can be regulated at the level of initiation of protein biosynthesis via structural elements present at the 5' untranslated region of mRNAs. These folded mRNA segments may bind to the ribosome, thus blocking translation until the mRNA unfolds. Here, we report a series of cryo-electron microscopy snapshots of ribosomal complexes directly visualizing either the mRNA structure blocked by repressor protein S15 or the unfolded, active mRNA. In the stalled state, the folded mRNA prevents the start codon from reaching the peptidyl-tRNA (P) site inside the ribosome. Upon repressor release, the mRNA unfolds and moves into the mRNA channel allowing translation initiation. A comparative structure and sequence analysis suggests the existence of a universal stand-by site on the ribosome (the 30S platform) dedicated for binding regulatory 5' mRNA elements. Different types of mRNA structures may be accommodated during translation preinitiation and regulate gene expression by transiently stalling the ribosome.     

Translocation makes the ribosome less compact

Translating ribosomes of Escherichia coli were prepared either in the pre-translocation or in the post-translocation states by a special technique based on the use of poly(U)-Sepharose columns where the template was coupled to the matrix through splittable -S-S- bridges. Elongation factors were absent from the final preparations. A neutron scattering study of the translating ribosomes in the two functional states was performed at different contrasts (various 1H2O/2H2O mixtures). Under conditions of a high contrast for the protein constituent the radius of gyration of the post-translocation-state ribosomes was found to be slightly greater than that of the pre-translocation-state ribosomes. Using the results of this study the conclusion can be drawn that translocation is accompanied by a spatial displacement of some parts of the ribosome with a magnitude of several ?ngstr?m units.     

Localization of 5' and 3' ends of the ribosome-bound segment of template polynucleotides by immune electron microscopy

Poly(U) with an average chain length of 40-70 nucleotides was modified at the 5'- or 3'-terminal residues with 2,4-dinitrophenyl derivatives. The modified poly(U) was used to form 30S.poly(U) or 70S.poly(U).Phe-tRNA complexes. Localization of the 5' and 3' ends of the template polynucleotide on the 30S subunit and the 70S ribosome was performed by immune electron microscopy using antibodies against dinitrophenyl haptens. The 5' and 3' ends of poly(U) (putative entry and exit sites of the message) were found in the same region both on the 30S subunit and the 70S ribosome. They were located on the dorsal side of the 30S subunit between the head and the body near the groove bordering the side ledge (platform). Comparison of the size of this region with the possible length of the polynucleotide chain covered by the ribosome allowed us to suggest that the message makes a 'U-turn" (or forms a 'loop') as it passes through the ribosome.     

Interactions of translational factor EF-G with the bacterial ribosome before and after mRNA translocation

A conserved translation factor, known as EF-G in bacteria, promotes the translocation of tRNA and mRNA in the ribosome during protein synthesis. Here, EF-G.ribosome complexes in two intermediate states, before and after mRNA translocation, have been probed with hydroxyl radicals generated from free Fe(II)-EDTA. Before mRNA translocation and GTP hydrolysis, EF-G protected a limited set of nucleotides in both subunits of the ribosome from cleavage by hydroxyl radicals. In this state, an extensive set of nucleotides, in the platform and head domains of the 30S subunit and in the L7/L12 stalk region of the 50S subunit, became more exposed to hydroxyl radical attack, suggestive of conformational changes in these domains. Following mRNA translocation, EF-G protected a larger set of nucleotides (23S rRNA helices H43, H44, H89, and H95; 16S rRNA helices h5 and h15). No nucleotide with enhanced reactivity to hydroxyl radicals was detected in this latter state. Both before and after mRNA translocation, EF-G protected identical nucleotides in h5 and h15 of the 30S subunit. These results suggest that h5 and h15 may remain associated with EF-G during the dynamic course of the translocation mechanism. Nucleotides in H43 and H44 of the 50S subunit were protected only after translocation and GTP hydrolysis, suggesting that these helices interact dynamically with EF-G. The effects in H95 suggest that EF-G interacts weakly with H95 before mRNA translocation and strongly and more extensively with this helix following mRNA translocation.     

Time of action of 4.5 S RNA in Escherichia coli translation

A new class of suppressor mutants helps to define the role of 4.5 S RNA in translation. The suppressors reduce the requirement for 4.5 S RNA by increasing the intracellular concentration of uncharged tRNA. Suppression probably occurs by prolonging the period in which translating ribosomes have translocated but not yet released the uncharged tRNA, indicating that this is the point at which 4.5 S RNA enters translation. The release of 4.5 S RNA from polysomes is affected by antibiotics that inhibit protein synthesis. The antibiotic-sensitivity of this release indicates that 4.5 S RNA exits the ribosome following translocation and prior to release of protein synthesis elongation factor G. These results indicate that 4.5 S RNA acts immediately after ribosomal translocation. A model is proposed in which 4.5 S RNA stabilizes the post-translocation state by replacing 23 S ribosomal RNA as a binding site for elongation factor G. The 4.5 S RNA-requirement of mutants altered in 23 S ribosomal RNA support this model.     

Measurement of internal movements within the 30 S ribosomal subunit using Förster resonance energy transfer

We have used F?rster resonance energy transfer (FRET) to study specific conformational changes in the Escherichia coli 30 S ribosomal subunit that occur upon association with the 50 S subunit. By measuring energy transfer between 13 different pairs of fluorescent probes attached to specific positions on 30 S subunit proteins, we have monitored changes in distance between different locations within the 30 S subunit in its free and 50 S-bound states. The measured distance changes provide restraints for modeling the movement that occurs within the 30 S subunit upon formation of the 70 S ribosome in solution. Treating the head, body, and platform domains of the 30 S subunit as simple rigid bodies, the lowest-energy solution converges on a model that satisfies each of the individual FRET restraints. In this model, the 30 S subunit head tilts towards the 50 S subunit, similar to the movement found in comparing 30 S subunits and 70 S ribosomes from X-ray and cryo-electron microscope structures, and the platform is predicted to undergo a clock-wise rotation upon association.