Post-translocation Complexes Research Articles

Conformational dynamics of eukaryotic translation.

The ribosome is a molecular machine that adopts multiple conformations during translation. The body of the small ribosomal subunit rotates relatively to the large ribosomal subunit by about 9 degrees. This movement reconfigures tRNA binding sites, which results in a partial movement of tRNAs on the ribosome. At the beginning of the elongation cycle, the ribosome awaits A-site decoding and occupies the non-rotated conformation. Upon A-site tRNA binding, the ribosome rapidly catalyzes peptidyl transfer. It is followed by counterclockwise rotations of the small ribosomal subunit that puts the ribosome into the rotated state. The resultant pre-translocation complex is dynamic and exchanges between rotated and non-rotated conformations. Eukaryotic elongation factor 2 (eEF2) catalyzes translocation, which moves tRNA-mRNA and resets the ribosome into the non-rotated state. Despite extensive biochemical and structural studies, the underlying mechanism of translation elongation remains elusive. We developed a single-molecule fluorescence resonance energy transfer (smFRET) system that allows the observation of intersubunit conformational change of yeast ribosomes in real-time. We followed the ribosome conformational change in pre- and post-translocation ribosome complexes. To interrogate the mechanism of translocation, we applied antifungal sordarin. Sordarin stabilizes eEF2 on the ribosome after GTP hydrolysis and phosphate release. Our results suggest that in the presence of sordarin, the mid and late stages of translocation are thermally driven.

Two alternative conformations of mRNA in the human ribosome during elongation and termination of translation as revealed by EPR spectroscopy

The conformation of mRNA in the region of the human 80S ribosome decoding site was monitored using 11-mer mRNA analogues that bore nitroxide spin labels attached to the terminal nucleotide bases. Intramolecular spin–spin distances were measured by DEER/PELDOR spectroscopy in model complexes mimicking different states of the 80S ribosome during elongation and termination of translation. The measurements revealed that in all studied complexes, mRNA exists in two alternative conformations, whose ratios are different in post-translocation, pre-translocation and termination complexes. We found that the presence of a tRNA molecule at the ribosomal A site decreases the relative share of the more extended mRNA conformation, whereas the binding of eRF1 (alone or in a complex with eRF3) results in the opposite effect. In the termination complexes, the ratios of mRNA conformations are practically the same, indicating that a part of mRNA bound in the ribosome channel does not undergo significant structural alterations in the course of completion of the translation. Our results contribute to the understanding of mRNA molecular dynamics in the mammalian ribosome channel during translation.

Extraction of mRNA from Stalled Ribosomes by the Ski Complex.

The evolutionarily conserved Ski2-Ski3-Ski8 (Ski) complex containing the 3'→5' RNA helicase Ski2 binds to 80S ribosomes near the mRNA entrance and facilitates 3'→5' exosomal degradation of mRNA during ribosome-associated mRNA surveillance pathways. Here, we assayed Ski's activity using an invitro reconstituted translation system and report that this complex efficiently extracts mRNA from 80S ribosomes in the 3'→5' direction in a nucleotide-by-nucleotide manner. The process is ATP dependent and can occur on pre- and post-translocation ribosomal complexes. The Ski complex can engage productively with mRNA and extract it from 80S complexes containing as few as 19 (but not 13) 3'-terminal mRNA nucleotides starting from the P site. The mRNA-extracting activity of the Ski complex suggests that its role in mRNA quality control pathways is not limited to acceleration of exosomal degradation and could include clearance of stalled ribosomes from mRNA, poising mRNA for degradation and rendering stalled ribosomes recyclable by Pelota/Hbs1/ABCE1.

Translocation kinetics and structural dynamics of ribosomes are modulated by the conformational plasticity of downstream pseudoknots.

Downstream stable mRNA secondary structures can stall elongating ribosomes by impeding the concerted movements of tRNAs and mRNA on the ribosome during translocation. The addition of a downstream mRNA structure, such as a stem-loop or a pseudoknot, is essential to induce -1 programmed ribosomal frameshifting (-1 PRF). Interestingly, previous studies revealed that -1 PRF efficiencies correlate with conformational plasticity of pseudoknots, defined as their propensity to form incompletely folded structures, rather than with the mechanical properties of pseudoknots. To elucidate the detailed molecular mechanisms of translocation and -1 PRF, we applied several smFRET assays to systematically examine how translocation rates and conformational dynamics of ribosomes were affected by different pseudoknots. Our results show that initial pseudoknot-unwinding significantly inhibits late-stage translocation and modulates conformational dynamics of ribosomal post-translocation complexes. The effects of pseudoknots on the structural dynamics of ribosomes strongly correlate with their abilities to induce -1 PRF. Our results lead us to propose a kinetic scheme for translocation which includes an initial power-stroke step and a following thermal-ratcheting step. This scheme provides mechanistic insights on how selective modulation of late-stage translocation by pseudoknots affects -1 PRF. Overall our findings advance current understanding of translocation and ribosome-induced mRNA structure unwinding.

Single-molecule FRET studies on interactions between elongation factor 4 (LepA) and ribosomes

Elongation factor 4 (EF4) is one of the highly conserved translational GTPases, whose functions are largely unknown. Structures of EF4 bound ribosomal PRE-translocation and POST-translocation complexes have both been visualized. On top of cellular, structural, and biochemical studies, several controversial models have been raised to rationalize functions of EF4. However, how EF4 modulates elongation through its interactions with ribosomes has not been revealed. Here, using single-molecule fluorescence resonance energy transfer assays, we directly captured short-lived EF4·GTP bound ribosomal PRE and POST translocation complexes, which may adopt slightly different conformations from structures prepared using GDP, GDPNP, or GDPCP. Furthermore, we revealed that EF4·GTP severely impairs delivery of aminoacyl-tRNA into the A-site of the ribosome and moderately accelerates translocation. We proposed that functions of EF4 are to slow overall elongation and to stall majority of ribosomes in POST states under stress conditions.

Functional Dynamics within the Human Ribosome Regulate the Rate of Active Protein Synthesis

The regulation of protein synthesis contributes to gene expression in both normal physiology and disease, yet kinetic investigations of the human translation mechanism are currently lacking. Using single-molecule fluorescence imaging methods, we have quantified the nature and timing of structural processes in human ribosomes during single-turnover and processive translation reactions. These measurements reveal that functional complexes exhibit dynamic behaviors and thermodynamic stabilities distinct from those observed for bacterial systems. Structurally defined sub-states of pre- and post-translocation complexes were sensitive to specific inhibitors of the eukaryotic ribosome, demonstrating the utility of this platform to probe drug mechanism. The application of three-color single-molecule fluorescence resonance energy transfer (smFRET) methods further revealed a long-distance allosteric coupling between distal tRNA binding sites within ribosomes bearing three tRNAs, which contributed to the rate of processive translation.

Model of EF4-induced ribosomal state transitions and mRNA translocation

EF4, a highly conserved protein present in bacteria, mitochondria and chloroplasts, can bind to both the posttranslocation and pretranslocation ribosomal complexes. When binding to the posttranslocation state, it catalyzes backward translocation to a pretranslocation state. When binding to the pretranslocation state, it catalyzes transition to another pretranslocation state that is similar and possibly identical to that resulting from the posttranslocation state bound by EF4, and competes with EF-G to regulate the elongation cycle. However, the molecular mechanism on how EF4 induces state transitions and mRNA translocation remains unclear. Here, we present both the model for state transitions induced by EF4 binding to the posttranslocation state and that by EF4 binding to the pretranslocation state, based on which we study the kinetics of EF4-induced state transitions and mRNA translocation, giving quantitative explanations of the available experimental data. Moreover, we present some predicted results on state transitions and mRNA translocation induced by EF4 binding to the pretranslocation state complexed with the mRNA containing a duplex region.

Monitoring Translation with Modified mRNAs Strategically Labeled with Isomorphic Fluorescent Guanosine Mimetics

Here we examine three mRNAs, site-specifically modified at codon positions 4, 5, and 6 with a new emissive and responsive isosteric guanosine mimetic ((th)G), with the goal of developing real time assays for monitoring translation-related events at nucleotide resolution. All three emissive mRNAs tested form initiation (70SIC), pretranslocation (PRE), and posttranslocation (POST) complexes. In most cases spectral differences are seen on binding of the mRNA to the ribosome during 70SIC formation and on conversion of 70SIC to PRE complexes and PRE complexes to POST complexes. These differences allow measurement of the kinetics of such conversions by changes in the fluorescence of labeled mRNAs. Such measurements directly identify a specific step during PRE complex formation, provisionally assigned to codon:anticodon-loop base pair formation, that follows aa-tRNA.EF-Tu.GTP ternary complex binding to the ribosome and precedes aa-tRNA accommodation into the A-site of the ribosome. These observations demonstrate not only the functionality of mRNAs modified with the emissive guanosine mimetic but also the potential this mimetic offers for observing the formation and disappearance of discrete intermediates during the polypeptide elongation cycle.

The conserved protein EF4 (LepA) modulates the elongation cycle of protein synthesis

EF4 (LepA), a strongly conserved protein, is important for bacterial growth and functional protein biosynthesis under certain conditions and is quite similar structurally to the translocase EF-G. The elongation cycle in protein synthesis is characterized by ribosome oscillation between pretranslocation (PRE) and posttranslocation (POST) complexes. Here, using ensemble single turnover and equilibrium experiments, as well as single molecule FRET measurements, we demonstrate that EF4 can compete with EF-G for binding to the PRE complex. Such EF4 binding results in formation of a complex, denoted X(3), that effectively sequesters a catalytically active ribosome, leading to a transient inhibition of elongation that provides a mechanism for optimization of functional protein synthesis. Earlier [Liu H, et al. (2010) J Mol Biol 396:1043-1052] we demonstrated that EF4 also reacts with POST complex, leading to the formation of a complex, I(3), that appears to be identical with X(3). Our present results strongly suggest that PRE complex is the principal target of EF4 action on translation, rather than POST complex as had been previously supposed.

Allosteric collaboration between elongation factor G and the ribosomal L1 stalk directs tRNA movements during translation

Determining the mechanism by which tRNAs rapidly and precisely transit through the ribosomal A, P, and E sites during translation remains a major goal in the study of protein synthesis. Here, we report the real-time dynamics of the L1 stalk, a structural element of the large ribosomal subunit that is implicated in directing tRNA movements during translation. Within pretranslocation ribosomal complexes, the L1 stalk exists in a dynamic equilibrium between open and closed conformations. Binding of elongation factor G (EF-G) shifts this equilibrium toward the closed conformation through one of at least two distinct kinetic mechanisms, where the identity of the P-site tRNA dictates the kinetic route that is taken. Within posttranslocation complexes, L1 stalk dynamics are dependent on the presence and identity of the E-site tRNA. Collectively, our data demonstrate that EF-G and the L1 stalk allosterically collaborate to direct tRNA translocation from the P to the E sites, and suggest a model for the release of E-site tRNA.

Structures of phi29 DNA polymerase complexed with substrate: the mechanism of translocation in B-family polymerases

Replicative DNA polymerases (DNAPs) move along template DNA in a processive manner. The structural basis of the mechanism of translocation has been better studied in the A-family of polymerases than in the B-family of replicative polymerases. To address this issue, we have determined the X-ray crystal structures of phi29 DNAP, a member of the protein-primed subgroup of the B-family of polymerases, complexed with primer-template DNA in the presence or absence of the incoming nucleoside triphosphate, the pre- and post-translocated states, respectively. Comparison of these structures reveals a mechanism of translocation that appears to be facilitated by the coordinated movement of two conserved tyrosine residues into the insertion site. This differs from the mechanism employed by the A-family polymerases, in which a conserved tyrosine moves into the templating and insertion sites during the translocation step. Polymerases from the two families also interact with downstream single-stranded template DNA in very different ways.

Effects of Nucleotides and Nucleotide Analogue Inhibitors of HIV-1 Reverse Transcriptase in a Ratchet Model of Polymerase Translocation

A single cycle of nucleotide incorporation by the reverse transcriptase of the human immunodeficiency virus type 1 (HIV-1 RT) involves the initial binding of an incoming nucleotide, a conformational change that traps the substrate, the formation of a new phosphodiester bond, the release of pyrophosphate (PPi), and ultimately polymerase translocation, which clears the nucleotide binding site. This article reviews different mechanistic models for polymerase translocation with emphasis placed on HIV-1 RT. Structure-function analyses of stalled complexes of HIV-1 RT provide strong evidence to suggest that the enzyme can oscillate between pre- and post-translocational states. Nucleotide hydrolysis is not required for the movement of the polymerase in a stalled configuration; thermal energy is sufficient to allow random bidirectional sliding. The next complementary nucleotide, following the incorporated chain-terminator, acts like a pawl of a ratchet that traps the enzyme in the post-translocation state and prevents the reverse movement. Quantitative footprinting experiments have shown that the concentration of the templated nucleotide required to shift the translocational equilibrium forward depends crucially on the structure of the 3'end of the primer. Changes in the relative population of pre- and post-translocation complexes can influence rates of excision of incorporated NRTIs, which, in turn, affects drug susceptibility. The concept of a ratchet model of HIV-1 RT translocation and its implications for drug action and resistance, and the discovery and development of novel antiviral compounds is discussed.

Scientists Share Nuclear Secrets at Jekyll Island

Scientists Share Nuclear Secrets at Jekyll Island

Interaction of Thiostrepton and Elongation Factor-G with the Ribosomal Protein L11-binding Domain

Ribosomal protein L11 and the L11 binding region of ribosomal RNA constitute an important domain involved in active functions of the ribosome during translation. We studied the effects of L11 knock-out and truncation mutations on the structure of the rRNA in this region and on its interactions with a translation elongation factor and the antibiotic thiostrepton. The results indicated that the structure of the L11-binding rRNA becomes conformationally flexible when ribosomes lack the entire L11 protein, but not when the C-terminal domain is present on ribosomes. Probing wild type and mutant ribosomes in the presence of the antibiotic thiostrepton and elongation factor-G (EF-G) rigorously localized the binding cleft of thiostrepton and suggested a role for the rRNA in the L11-binding domain in modulating factor binding. Our results also provide evidence that the structure of the rRNA stabilized by the C-terminal domain of L11 is necessary to stabilize EF-G binding in the post-translocation state, and thiostrepton may modulate this structure in a manner that interferes with the ribosome-EF-G interaction. The implications for recent models of thiostrepton activity and factor interactions are discussed.

Alternating translocation of protein substrates from both ends of ClpXP protease.

In ClpXP protease complexes, hexameric rings of the ATP-dependent ClpX chaperone stack on one or both faces of the double-heptameric rings of ClpP. We used electron microscopy to record the initial binding of protein substrates to ClpXP and their accumulation inside proteolytically inactive ClpP. Proteins with N- or C-terminal recognition motifs bound to complexes at the distal surface of ClpX and, upon addition of ATP, were translocated to ClpP. With a partially translocated substrate, the non-translocated portion remained on the surface of ClpX, aligned with the central axis of the complex, confirming that translocation proceeds through the axial channel of ClpXP. Starting with substrate bound on both ends, most complexes translocated substrate from only one end, and rarely (<5%) from both ends. We propose that translocation from one side is favored for two reasons: initiation of translocation is infrequent, making the probability of simultaneous initiation low; and, further, the presence of protein within the cis side translocation channel or within ClpP generates an inhibitory signal blocking translocation from the trans side.

Euplotes telomerase: evidence for limited base-pairing during primer elongation and dGTP as an effector of translocation.

The telomeric sequence repeats at the ends of eukaryotic chromosomes are maintained by the ribonucleoprotein enzyme telomerase. Telomeric DNA primers are bound by telomerase both at the active site, which includes base-pairing with the RNA template, and at a second anchor site. The stabilities of Euplotes aediculatus primer-telomerase complexes were determined by measuring their dissociation rates (koff), using an assay involving photo-cross-linking at the anchor site. The primer length was varied, and mismatched substitutions were introduced in a systematic manner. We observed that koff does not scale with primer length as expected for accumulated primer-template base-pairing. This suggests that telomerase maintains a more-or-less constant number of base pairs, similar to the transcription bubble maintained by RNA polymerase. An upper limit was estimated by comparing the experimental koff for the primer-telomerase complex to that of a model DNA-RNA duplex. All the binding energy could be attributed to 10 or 11 base pairs; alternatively, there could be <10 base pairs, with the remaining energy contributed by other parts of telomerase. Most primers exhibited biphasic dissociation kinetics, with variations in both the amount in each phase and the rate for each phase. Since the cross-links monitored in the dissociation assay were all formed with the 5' region of the primer, the two phases may arise from different base-pairing registers with the RNA template, possibly representing pre- and post-translocation complexes. A shift from slow phase to fast phase dissociation was observed in the presence of dGTP, which may implicate dGTP as a positive effector of translocation.

Structural dynamics of translating ribosomes: 16S ribosomal RNA bases that may move twice during translocation.

Recent footprinting, sedimentation and neutron-scattering results obtained in vivo or on pre-translocation and post-translocation ribosomal complexes are integrated with cross-linking and immunoelectron microscopy information. It is proposed that the 30S subunit pulses during translocation and that its pre- and post-translocation structures are not necessarily identical. Accordingly, translocation is characterized by three consecutive conformational states of the 30S and 50S subunits. State 1 (the pre-translocation state) lasts until the elongation factor EF-G.GTP complex binds to the ribosome or adopts the GTPase conformation. State 2 (the translocation state, or the peak or plateau of the pulse) follows and lasts until EF-G adopts a subsequent conformation or is released from the ribosome. State 3 (the post-translocation state) ensues and lasts until A (aminoacyl) site binding of aminoacyl-tRNA. In state 2, 16S RNA hairpins 26 and 33-33A, located in the platform and the head of the 30S subunit, respectively, become kinked or twisted, and residue A1503, near the decoding site, becomes exposed. A platform twist is associated with P (peptide) to E (exit) site tRNA movements and a head twist with pivoting of the peptidyl-tRNA elbow from the A to the P site, around a (retractable?) S19 domain. These twists result in an unlocking of the platform and the head from the 50S subunit. Exposure of A1503 is tentatively associated with movements of mRNA or tRNA anticodon stem-loops. These twisted or otherwise-exposed residues readopt their previous setting upon completion of translocation, i.e. states 1 and 3 of 16S RNA differ more from state 2 than from each other.(ABSTRACT TRUNCATED AT 250 WORDS)

Reduced ribosomal binding of eukaryotic elongation factor 2 following ADP-ribosylation. Difference in binding selectivity between polyribosomes and reconstituted monoribosomes

The biological activity of elongation factor 2 (EF-2) following NAD +- and diphtheria-toxin-dependent ADP-ribosylation was studied (i) in translation experiments using the reticulocyte lysate system and (ii) in ribosomal binding experiments using either reconstituted empty rat liver ribosomes or programmed reticulocyte polysomes. Treatment of the lysates with toxin and NAD + at a NAD + ribosome ratio of 4 resulted in a 90% inhibition of the amino acid incorporation rate. The inhibition was overcome by the addition of native EF-2. At this level of inhibition more than 90% of the EF-2 present in the lysates was ADP-ribosylated and the total ribosome association of EF-2 was reduced by approx. 50%. All of the remaining unmodified factor molecules were associated with the ribosomes, whereas only about 3% of the ribosylated factor was ribosome-associated. The nucleotide requirement for the binding of EF-2 to empty reconstituted rat liver ribosomes and programmed reticulocyte polysomes was studied together with the stability of the resulting EF-2· ribosome complexes using purified 125I-labelled rat liver EF-2. With both types of ribosomes, the complex formation was strictly nucleotide-dependent. Stable, high-affinity complexes were formed in the presence of the non-hydrolysable GTP analogue guanosine 5′-(β,γ-methylene)triphosphate (Guo PP[CH 2] P). In contrast to the reconstituted ribosomes, GTP stimulated the formation of high-affinity complexes in the presence of polysomes, albeit at a lower efficiency than Guo PP[CH 2] P. The formation of high-affinity complexes was restricted to polysomes in the pretranslocation phase of the elongation cycle. Low-affinity post-translocation complexes, demonstrable after fixation, were formed in the presence of GTP, Guo PP[CH 2] P and GDP. In polysomes, these complexes involved a different population of particles than did the high-affinity complexes. In the binding experiments using reconstituted or programmed ribosomes, the pretranslocation binding of EF-2 observed in the presence of Guo PP[CH 2] P was reduced by approx. 50% after ADP-ribosylation, whereas the post-translocation binding in the presence of GDP was unaltered. The data indicate that the inhibition of translocation caused by diphtheria toxin and NAD + is mediated through a reduced affinity of the ADP-ribosylated EF-2 for binding to ribosomes in the pretranslocation state.

Apparent changes in ribosome conformation during protein synthesis. Centrifugation at high speed distorts initiation, pretranslocaton, and posttranslocation complexes to a different extent.

Apparent changes in ribosome conformation during protein synthesis. Centrifugation at high speed distorts initiation, pretranslocaton, and posttranslocation complexes to a different extent.