Ribosomal Translocation Research Articles

Muscarinic acetylcholine receptors regulate the dephosphorylation of eukaryotic translation elongation factor 2 in SNU-407 colon cancer cells

Previously, we showed that muscarinic acetylcholine receptors (mAChRs) promote global protein biosynthesis in SNU-407 colon cancer cells. However, the molecular mechanisms underlying this event are poorly understood. Here, we asked whether mAChRs modulate the activity of eukaryotic translation elongation factor 2 (eEF2), which controls ribosomal translocation during the peptide elongation step. When SNU-407 cells were treated with the cholinergic agonist carbachol, eEF2 phosphorylation at T56 was decreased in a dose- and time-dependent manner. The muscarinic antagonist atropine almost completely blocked this effect of carbachol, demonstrating that mAChRs specifically regulate eEF2 dephosphorylation. We also investigated the signaling pathways that connect mAChR stimulation to eEF2 dephosphorylation using chemical inhibitors. Treating cells with U0126, a potent MEK1/2 inhibitor, decreased carbachol-stimulated eEF2 dephosphorylation. In contrast, the mTORC1 inhibitor rapamycin did not have a significant effect on eEF2 dephosphorylation. We also found that the protein kinase C (PKC) inhibitor GF109203X substantially reduced eEF2 dephosphorylation. Together, our experimental data indicate that the MEK1/2-ERK1/2 pathway and the PKC pathway, but not the mTORC1-S6K1 pathway, are involved in mAChR-mediated eEF2 dephosphorylation.

The uncharacterized bacterial protein YejG has the same architecture as domain III of elongation factor G.

InterPro family IPR020489 comprises ~1000 uncharacterized bacterial proteins. Previously we showed that overexpressing the Escherichia coli representative of this family, EcYejG, conferred low-level resistance to aminoglycoside antibiotics. In an attempt to shed light on the biochemical function of EcYejG, we have solved its structure using multinuclear solution NMR spectroscopy. The structure most closely resembles that of domain III from elongation factor G (EF-G). EF-G catalyzes ribosomal translocation and mutations in EF-G have also been associated with aminoglycoside resistance. While we were unable to demonstrate a direct interaction between EcYejG and the ribosome, the protein might play a role in translation.

Spontaneous ribosomal translocation of mRNA and tRNAs into a chimeric hybrid state

The elongation factor G (EF-G)-catalyzed translocation of mRNA and tRNA through the ribosome is essential for vacating the ribosomal A site for the next incoming aminoacyl-tRNA, while precisely maintaining the translational reading frame. Here, the 3.2-Å crystal structure of a ribosome translocation intermediate complex containing mRNA and two tRNAs, formed in the absence of EF-G or GTP, provides insight into the respective roles of EF-G and the ribosome in translocation. Unexpectedly, the head domain of the 30S subunit is rotated by 21°, creating a ribosomal conformation closely resembling the two-tRNA chimeric hybrid state that was previously observed only in the presence of bound EF-G. The two tRNAs have moved spontaneously from their A/A and P/P binding states into ap/P and pe/E states, in which their anticodon loops are bound between the 30S body domain and its rotated head domain, while their acceptor ends have moved fully into the 50S P and E sites, respectively. Remarkably, the A-site tRNA translocates fully into the classical P-site position. Although the mRNA also undergoes movement, codon-anticodon interaction is disrupted in the absence of EF-G, resulting in slippage of the translational reading frame. We conclude that, although movement of both tRNAs and mRNA (along with rotation of the 30S head domain) can occur in the absence of EF-G and GTP, EF-G is essential for enforcing coupled movement of the tRNAs and their mRNA codons to maintain the reading frame.

Measuring the Mechanical Forces during Ribosome Translocation via Ef-G Crosslinking

Measuring the Mechanical Forces during Ribosome Translocation via Ef-G Crosslinking

A Force Method to Study the EF-G Mechanism during the Ribosome Translocation and Frameshifting

A Force Method to Study the EF-G Mechanism during the Ribosome Translocation and Frameshifting

Abstract 57: Exosomes Derived From Ischemic Cerebral Endothelial Cells Promote Axonal Growth

Axonal remodeling is a critical aspect of spontaneous brain repair after stroke. Stroke activates cerebral endothelial cells (CECs) in ischemic blood vessels. Communications between activated CECs and axonal remodeling in ischemic brain have not been fully investigated. Exosomes play vital roles in intercellular communication. We investigated the effect of exosomes derived from CECs harvested from vessels of ischemic rat brains (isCEC-exos) on axonal growth of cortical neurons. Application of isCEC-exos into axons of the neurons cultured in a microfluidic device which separates axons from their somata resulted in significant (p<0.05, n=45 axons) increases of axonal growth (982±24 μm) compared to CEC-exos derived from non-ischemic vessels (nCEC-exos, 793±19 μm). nCEC-exos significantly enhanced axonal growth compared to non-treatment (567±14 μm). Axonal internalization of CEC-exos was confirmed by tracking GFP-exosomes. qRT-PCR and miRNA PCR array analysis revealed that compared to nCEC-exos, >50 mature miRNAs were enriched within isCEC-exos, including miR-19a. Axonal application of isCEC-exos elevated these miRNAs and their pre-miRNAs in axons and somata (from 21 to 85 folds, n=3, p<0.01) compared to non-treated group. Because CEC-exos do not contain the precursor form of miRNAs, these data suggest that CEC-exos transfer their mature miRNAs into distal axons, which triggered increases in precursor and mature miRNAs in distal axons and somata. Moreover, compared to the non-treated group, Western blot analysis (n=3/group) showed that axonal application of isCEC-exos locally reduced axonal growth inhibitory proteins, PTEN (~90%), Sema6A (~75%), and RhoA (~99%), which was inversely associated with increased miRNAs that target these proteins. Furthermore, transient blockage of ribosome translocation with emetine, a protein synthesis inhibitor, in axons abolished the effect of isCEC-exos on distal axonal growth and on alteration of miRNAs and their target proteins. Collectively, our data demonstrate that isCEC-exos internalized by distal axons facilitate axonal growth by altering miRNAs and their target protein profiles in recipient neurons, and these exosomes may potentially provide a therapeutic means to enhance axonal growth.

Increased Calcium Influx through L-type Calcium Channels in Human and Mouse Neural Progenitors Lacking Fragile X Mental Retardation Protein.

SummaryThe absence of FMR1 protein (FMRP) causes fragile X syndrome (FXS) and disturbed FMRP function is implicated in several forms of human psychopathology. We show that intracellular calcium responses to depolarization are augmented in neural progenitors derived from human induced pluripotent stem cells and mouse brain with FXS. Increased calcium influx via nifedipine-sensitive voltage-gated calcium (Cav) channels contributes to the exaggerated responses to depolarization and type 1 metabotropic glutamate receptor activation. The ratio of L-type/T-type Cav channel expression is increased in FXS progenitors and correlates with enhanced progenitor differentiation to glutamate-responsive cells. Genetic reduction of brain-derived neurotrophic factor in FXS mouse progenitors diminishes the expression of Cav channels and activity-dependent responses, which are associated with increased phosphorylation of the phospholipase C-γ1 site within TrkB receptors and changes of differentiating progenitor subpopulations. Our results show developmental effects of increased calcium influx via L-type Cav channels in FXS neural progenitors.

Dual DNA rulers reveal an ‘mRNA looping’ intermediate state during ribosome translocation

ABSTRACTThe precise 3-nucleotide movement of mRNA is critical for translation fidelity. One mRNA translocation error propagates to all of the following codons, which is detrimental to the cell. However, none of the current methods can reveal the mRNA dynamics near the ribosome entry site, which limits the understanding of this important issue. We have developed an assay of dual DNA rulers that provides such capability. By uniquely probing both the 3ʹ- and 5ʹ-ends of mRNA, we observed an antibiotic-trapped intermediate state that is consistent with a ribosomal conformation containing mRNA asymmetric partial displacements at its entry and exit sites. Based on the available ribosome structures and computational simulations, we proposed a ‘looped’ mRNA conformation, which suggested a stepwise ‘inchworm’ mechanism for ribosomal translocation. The same ‘looped’ intermediate state identified with the dual rulers persists with a ‘-1’ frameshifting motif, indicating that the branching point of normal and frameshifting translocations occurs at a later stage of translocation.

Ras Suppresses TXNIP Expression by Restricting Ribosome Translocation.

Oncogenic Ras upregulates aerobic glycolysis to meet the bioenergetic and biosynthetic demands of rapidly growing cells. In contrast, thioredoxin-interacting protein (TXNIP) is a potent inhibitor of glucose uptake and is frequently downregulated in human cancers. Our laboratory previously discovered that Ras activation suppresses TXNIP transcription and translation. In this study, we developed a system to study how Ras affects TXNIP translation in the absence of transcriptional effects. We show that whereas Ras drives a global increase in protein translation, it suppresses TXNIP protein synthesis by reducing the rate at which ribosomes transit the coding region of TXNIP mRNA. To investigate the underlying mechanism(s), we randomized or optimized the codons in the TXNIP message without altering the TXNIP primary amino acid sequence. Translation from these mRNA variants was still repressed by Ras, implying that mRNA secondary structure, microRNAs (miRNAs), RNA binding proteins, or codon usage does not contribute to the blockade of TXNIP synthesis. Rather, we show that the N terminus of the growing TXNIP polypeptide is the target for Ras-dependent translational repression. Our work demonstrates how Ras suppresses TXNIP translation elongation in the face of a global upregulation of protein synthesis and provides new insight into Ras-dependent metabolic reprogramming.

Eukaryotic translation elongation factor 2 (eEF2) catalyzes reverse translocation of the eukaryotic ribosome

During protein synthesis, a ribosome moves along the mRNA template and, using aminoacyl-tRNAs, decodes the template nucleotide triplets to assemble a protein amino acid sequence. This movement is accompanied by shifting of mRNA-tRNA complexes within the ribosome in a process called translocation. In living cells, this process proceeds in a unidirectional manner, bringing the ribosome to the 3' end of mRNA, and is catalyzed by the GTPase translation elongation factor 2 (EF-G in prokaryotes and eEF2 in eukaryotes). Interestingly, the possibility of spontaneous backward translocation has been shown in vitro for bacterial ribosomes, suggesting a potential reversibility of this reaction. However, this possibility has not yet been tested for eukaryotic ribosomes. Here, using a reconstituted mammalian translation system, we show that the eukaryotic elongation factor eEF2 catalyzes ribosomal reverse translocation at one mRNA triplet. We found that this process requires a cognate tRNA in the ribosomal E-site and cannot occur spontaneously without eEF2. The efficiency of this reaction depended on the concentrations of eEF2 and cognate tRNAs and increased in the presence of nonhydrolyzable GTP analogues. Of note, ADP-ribosylation of eEF2 domain IV blocked reverse translocation, suggesting a crucial role of interactions of this domain with the ribosome for the catalysis of the reaction. In summary, our findings indicate that eEF2 is able to induce ribosomal translocation in forward and backward directions, highlighting the universal mechanism of tRNA-mRNA movements within the ribosome.

Exaggerated Swivel Motions of the Small Subunit Head Domain are Required for tRNA Translocation through the Bacterial Ribosome

Translocation of tRNA through the ribosome is essential for protein synthesis and for maintaining the open reading frame of mRNA translation. Translocation is associated with two large-scale rearrangements within the ribosome: an intersubunit rotation of the small subunit relative to the large subunit (rotation); and an intrasubunit rotation of small subunit head relative to its body (head swivel). Recent single-molecule FRET studies (smFRET) suggested that exaggerated head swivel motions accompany the translocation reaction coordinate. Here, we integrate molecular dynamics simulations of the 70S ribosome translocation complex with single molecule FRET studies to more fully characterize the conformational changes in the ribosome accompanying translocation, and the transit of key intermediates that have recently been identified by structural and smFRET investigations. By explicitly incorporating FRET labels into the simulations, we obtain configurations of the ribosome highly consistent with measured FRET efficiencies for each translocation intermediate. We find that a larger amplitude head swivel configuration (‘super swivel’) satisfies the constraints of FRET efficiency and conformational changes predicted to accompany translocation. In so doing, our study supports a simplified translocation pathway consistent with extant structural data and the order of events during and the geometric constraints predicted by recent smFRET initiatives. Our simulations also characterize the influence of dye flexibility on the smFRET distance estimates.

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.

An Update on Sec61 Channel Functions, Mechanisms, and Related Diseases.

The membrane of the endoplasmic reticulum (ER) of nucleated human cells harbors the protein translocon, which facilitates membrane integration or translocation of almost every newly synthesized polypeptide targeted to organelles of the endo- and exocytotic pathway. The translocon comprises the polypeptide-conducting Sec61 channel and several additional proteins and complexes that are permanently or transiently associated with the heterotrimeric Sec61 complex. This ensemble of proteins facilitates ER targeting of precursor polypeptides, modification of precursor polypeptides in transit through the Sec61 complex, and Sec61 channel gating, i.e., dynamic regulation of the pore forming subunit to mediate precursor transport and calcium efflux. Recently, cryoelectron tomography of translocons in native ER membrane vesicles, derived from human cell lines or patient fibroblasts, and even intact cells has given unprecedented insights into the architecture and dynamics of the native translocon and the Sec61 channel. These structural data are discussed in light of different Sec61 channel activities including ribosome receptor function, membrane insertion, and translocation of newly synthesized polypeptides as well as the putative physiological roles of the Sec61 channel as a passive ER calcium leak channel. Furthermore, the structural insights into the Sec61 channel are incorporated into an overview and update on Sec61 channel-related diseases—the Sec61 channelopathies—and novel therapeutic concepts for their treatment.

Ribosomal protein L7/L12 is required for GTPase translation factors EF‐G, RF3, and IF2 to bind in their GTP state to 70S ribosomes

Ribosomal protein L7/L12 is associated with translation initiation, elongation, and termination by the 70S ribosome. The guanosine 5' triphosphate hydrolase (GTPase) activity of elongation factor G (EF-G) requires the presence of L7/L12, which is critical for ribosomal translocation. Here, we have developed new methods for the complete depletion of L7/L12 from Escherichia coli 70S ribosomes to analyze the effect of L7/L12 on the activities of the GTPase factors EF-G, RF3, IF2, and LepA. Upon removal of L7/L12 from ribosomes, the GTPase activities of EF-G, RF3, and IF2 decreased to basal levels, while the activity of LepA decreased marginally. Upon reconstitution of ribosomes with recombinant L12, the GTPase activities of all GTPases returned to full activity. Moreover, ribosome binding assays indicated that EF-G, RF3, and IF2 require L7/L12 for stable binding in the GTP state, and LepA retained > 50% binding. Lastly, an EF-G∆G' truncation mutant possessed ribosome-dependent GTPase activity, which was insensitive to L7/L12. Our results indicate that L7/L12 is required for stable binding of ribosome-dependent GTPases that harbor direct interactions to the L7/L12 C-terminal domains, either through a G' domain (EF-G, RF3) or a unique N-terminal domain (IF2). Furthermore, we hypothesize this interaction is concomitant with counterclockwise ribosomal intersubunit rotation, which is required for translocation, initiation, and post-termination.

Simulating Movement of the Ribosome Head during Translocation

Intrasubunit head movement has been identified as an essential motion required for translocation of mRNA through the ribosome. Recent single molecule FRET data has suggested that exaggerated motions of the head, beyond what are observed in structural studies, are required for the rate-limiting step of translocation. We use molecular simulations of the 70S ribosome translocation complexes to explore these exaggerated motions and identify regions of the ribosome that place constraints on the maximal displacements of the head.

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

Ribosomal translocation catalyzed by EF-G hydrolyzing GTP entails multiple conformational changes of ribosome and positional changes of tRNAs and mRNA in the ribosome. However, the detailed dynamic relations among these changes and EF-G sampling are not clear. Here, based on our proposed pathway of ribosomal translocation, we study theoretically the dynamic relations among these changes exhibited in the single molecule data and those exhibited in the ensemble kinetic data. It is shown that the timing of these changes in the single molecule data and that in the ensemble kinetic data show very different. The theoretical results are in agreement with both the available ensemble kinetic experimental data and the single molecule experimental data.

A novel translational control mechanism involving RNA structures within coding sequences.

The impact of RNA structures in coding sequences (CDS) within mRNAs is poorly understood. Here, we identify a novel and highly conserved mechanism of translational control involving RNA structures within coding sequences and the DEAD-box helicase Dhh1. Using yeast genetics and genome-wide ribosome profiling analyses, we show that this mechanism, initially derived from studies of the Brome Mosaic virus RNA genome, extends to yeast and human mRNAs highly enriched in membrane and secreted proteins. All Dhh1-dependent mRNAs, viral and cellular, share key common features. First, they contain long and highly structured CDSs, including a region located around nucleotide 70 after the translation initiation site; second, they are directly bound by Dhh1 with a specific binding distribution; and third, complementary experimental approaches suggest that they are activated by Dhh1 at the translation initiation step. Our results show that ribosome translocation is not the only unwinding force of CDS and uncover a novel layer of translational control that involves RNA helicases and RNA folding within CDS providing novel opportunities for regulation of membrane and secretome proteins.

Maintenance of Transcription-Translation Coupling by Elongation Factor P.

ABSTRACTUnder conditions of tight coupling between translation and transcription, the ribosome enables synthesis of full-length mRNAs by preventing both formation of intrinsic terminator hairpins and loading of the transcription termination factor Rho. While previous studies have focused on transcription factors, we investigated the role of Escherichia coli elongation factor P (EF-P), an elongation factor required for efficient translation of mRNAs containing consecutive proline codons, in maintaining coupled translation and transcription. In the absence of EF-P, the presence of Rho utilization (rut) sites led to an ~30-fold decrease in translation of polyproline-encoding mRNAs. Coexpression of the Rho inhibitor Psu fully restored translation. EF-P was also shown to inhibit premature termination during synthesis and translation of mRNAs encoding intrinsic terminators. The effects of EF-P loss on expression of polyproline mRNAs were augmented by a substitution in RNA polymerase that accelerates transcription. Analyses of previously reported ribosome profiling and global proteomic data identified several candidate gene clusters where EF-P could act to prevent premature transcription termination. In vivo probing allowed detection of some predicted premature termination products in the absence of EF-P. Our findings support a model in which EF-P maintains coupling of translation and transcription by decreasing ribosome stalling at polyproline motifs. Other regulators that facilitate ribosome translocation through roadblocks to prevent premature transcription termination upon uncoupling remain to be identified.

On the pathway of ribosomal translocation

The translocation of tRNAs coupled with mRNA in the ribosome is a critical process in the elongation cycle of protein synthesis. The translocation entails large-scale conformational changes of the ribosome and involves several intermediate states with tRNAs in different positions with respect to 30S and 50S ribosomal subunits. However, the detailed role of the intermediate states is unknown and the detailed mechanism and pathway of translocation is unclear. Here based on previous structural, biochemical and single-molecule data we present a translocation pathway by incorporating several intermediate states. With the pathway, we study theoretically (i) the kinetics of 30S head rotation associated with translocation catalyzed by wild-type EF-G, (ii) the dynamics of fluctuations between different tRNA states during translocation interfered with EF-G mutants and translocation-specific antibiotics, (iii) the kinetics of tRNA movement in 50S subunit and mRNA movement in 30S subunit in the presence of wild-type EF-G, EF-G mutants and translocation-specific antibiotics, (iv) the dynamics of EF-G sampling to the ribosome during translocation, etc., providing consistent and quantitative explanations of various available biochemical and single-molecule experimental data published in the literature. Moreover, we study the kinetics of 30S head rotation in the presence of EF-G mutants, providing predicted results. These have significant implications for the molecular mechanism and pathway of ribosomal translocation.

Stability of HIV Frameshift Site RNA Correlates with Frameshift Efficiency and Decreased Virus Infectivity.

Human immunodeficiency virus (HIV) replication is strongly dependent upon a programmed ribosomal frameshift. Here we investigate the relationships between the thermodynamic stability of the HIV type 1 (HIV-1) RNA frameshift site stem-loop, frameshift efficiency, and infectivity, using pseudotyped HIV-1 and HEK293T cells. The data reveal a strong correlation between frameshift efficiency and local, but not overall, RNA thermodynamic stability. Mutations that modestly increase the local stability of the frameshift site RNA stem-loop structure increase frameshift efficiency 2-fold to 3-fold in cells. Thus, frameshift efficiency is determined by the strength of the thermodynamic barrier encountered by the ribosome. These data agree with previous in vitro measurements, suggesting that there are no virus- or host-specific factors that modulate frameshifting. The data also indicate that there are no sequence-specific requirements for the frameshift site stem-loop. A linear correlation between Gag-polymerase (Gag-Pol) levels in cells and levels in virions supports the idea of a stochastic virion assembly mechanism. We further demonstrate that the surrounding genomic RNA secondary structure influences frameshift efficiency and that a mutation that commonly arises in response to protease inhibitor therapy creates a functional but inefficient secondary slippery site. Finally, HIV-1 mutants with enhanced frameshift efficiencies are significantly less infectious, suggesting that compounds that increase frameshift efficiency by as little as 2-fold may be effective at suppressing HIV-1 replication. HIV, like many retroviruses, utilizes a -1 programmed ribosomal frameshift to generate viral enzymes in the form of a Gag-Pol polyprotein precursor. Thus, frameshifting is essential for viral replication. Here, we utilized a panel of mutant HIV strains to demonstrate that in cells, frameshifting efficiency is correlated with the stability of the local thermodynamic barrier to ribosomal translocation. Increasing the stability of the frameshift site RNA increases the frameshift efficiency 2-fold to 3-fold. Mutant viruses with increased frameshift efficiencies have significantly reduced infectivity. These data suggest that this effect might be exploited in the development of novel antiviral strategies.