Circadian clocks are modulated by compartmentalized oscillating translation

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Three distinct peptides from the N domain of translation termination factor eRF1 surround stop codon in the ribosome

To study positioning of the polypeptide release factor eRF1 toward a stop signal in the ribosomal decoding site, we applied photoactivatable mRNA analogs, derivatives of oligoribonucleotides. The human eRF1 peptides cross-linked to these short mRNAs were identified. Cross-linkers on the guanines at the second, third, and fourth stop signal positions modified fragment 31–33, and to lesser extent amino acids within region 121–131 (the “YxCxxxF loop”) in the N domain. Hence, both regions are involved in the recognition of the purines. A cross-linker at the first uridine of the stop codon modifies Val66 near the NIKS loop (positions 61–64), and this region is important for recognition of the first uridine of stop codons. Since the N domain distinct regions of eRF1 are involved in a stop-codon decoding, the eRF1 decoding site is discontinuous and is not of “protein anticodon” type. By molecular modeling, the eRF1 molecule can be fitted to the A site proximal to the P-site-bound tRNA and to a stop codon in mRNA via a large conformational change to one of its three domains. In the simulated eRF1 conformation, the YxCxxxF motif and positions 31–33 are very close to a stop codon, which becomes also proximal to several parts of the C domain. Thus, in the A-site-bound state, the eRF1 conformation significantly differs from those in crystals and solution. The model suggested for eRF1 conformation in the ribosomal A site and cross-linking data are compatible.     

Pit2 assemblies at the cell surface are modulated by extracellular inorganic phosphate concentration

Pit2 is a type III sodium-dependent phosphate transporter and the cell surface receptor for amphotropic murine leukemia virus. Indirect arguments have previously suggested that retrovirus receptor assembly play a role in triggering membrane fusion. Using CHO cells expressing physiological amounts of functional versions of human Pit2 fused to various tagging epitopes, we provide evidence that Pit2 forms assemblies at the cell surface. Living cells were exposed to cross-linking reagents and protein extracts were treated with trifluoroacetic acid (TFA), a chemical that destroys all protein interactions but covalent links. Assemblies were also detected in the absence of cross-linking and TFA treatment, indicating that they are partially resistant to detergent denaturation. The formation of homo-oligomers was documented by the coimmunoprecipitation of differently tagged molecules. The amounts of Pit2 assemblies detected in the presence or in the absence of cross-linking reagents varied with extracellular inorganic phosphate concentration ([P(i)]). Variation of signal intensity was in the range of twofold, occurred in the absence of de novo protein synthesis and took place at the cell surface. These results indicate that Pit2 assemblies exhibit variable conformations at the surface of living cells. Susceptibility to virus infection and phosphate uptake also vary with extracellular [P(i)]. A model is proposed in which cell surface Pit2 assemblies switch from a compacted to an expanded configuration in response to changes of extracellular [P(i)], and possible relationships with the variation of biological activities are discussed.     

Dependence of the quality and the in vitro translation capacity of pine ribosome assemblies on the isolation procedure

The in vitro translation capacity of total ribosome assemblies isolated from the vegetative buds of small Scots pine (Pinus sylvestris L.) plants depends on the isolation procedure. Good yields and high values for protein synthesis were obtained in experiments in which polyvinyl pyrrolidone (PVP) was added to the grinding buffer. The polysome profiles obtained after sucrose density gradient centrifugation indicated the presence of polysomes in all samples. In addition, large ribosome aggregates were visible in the scanning electron micrographs. The use of an RNase inhibitor (RNasin) together with PVP did not improve the results, and treatment with ribonuclease (RNase, EC 3.1.27.5) destroyed the ability to synthesize protein. D, L-Dithiothreitol (DTT) and mercaptoethanol, if used instead of or together with PVP, gave low yields and also DTT destroyed the in vitro translation capacity of the ribosome assemblies. The polysome profiles had a high peak indicating dimers and often a descending series of peaks indicating polymers. A study of the scanning electron micrographs gave the impression that the largest polymers and aggregates had broken down. Protease K (EC 3.4.21.14) when added to the grinding buffer also destroyed the ability of the ribosomes to maintain protein synthesis in vitro. In this case, the shape of the polysome profiles gave the impression of successful isolation. Clumps of ribosomes, presumably originating from large aggregates, were visible in the scanning electron micrographs. Triton X-100 and 0.25 M NaCl in the grinding buffer extracted chromatin, which affected the results. The material lost during the extraction and purification processes consisted mainly of monosomes and their sub-units. On the basis of the above results it was concluded that the preservation of large polysomes and ribosome aggregates in the isolated ribosome assemblies is necessary if they are to maintain a high translation capacity. The content of the assemblies was best revealed in the scanning electron micrographs. The shape of the polysome profiles did not always correlate with the ability of the isolated ribosomes to synthesize proteins.     

Hydrogen-bonding assemblies constructed from metalloligand building blocks and H2O

New hydrogen-bonding assemblies were synthesized from the reaction of a metalloligand, [Cu(2,4-pydca)₂]²⁻ (L^Cu) (2,4-pydca = 2,4-pyridinedicarboxylate), with a Fe^II ion or an imidazole in an aqueous medium and crystallographically characterized. The obtained compounds, [Fe(H₂O)₆][Cu(2,4-pydca)₂] (1) and [Cu(2,4-pydca)(imidazole)₂] · 2H₂O (2), have metalloligand dimer units, [Cu₂(2,4-pydca)₄]⁴⁻ and [Cu₂(2,4-pydca)₂(imidazole)₄], respectively, each of which assembles by π-π (1) and hydrogen-bonding (2) interactions to form 1-D metalloligand arrays. The 1-D metalloligand arrays are linked by rich hydrogen-bonding interactions via H₂O molecules.     

Optimal conditions for translation by thylakoid-bound polysomes from pea chloroplasts

Polysomes bound to washed thylakoids from pea Pisum sativum cv Progress No. 9 chloroplasts are capable of protein synthesis when supplemented with amino acids, ATP and a regenerating system, GTP, and soluble factors required for translation. The extent of protein synthesis in previous reports, however, was quite low when compared to in organello translation. By systematic testing of parameters in the isolation of thylakoids and reaction mixture components we have been able to establish more optimal conditions. Incorporation of 2 to 10 nanomoles of leucine per milligram chlorophyll in a 20-minute reaction period is now possible, representing a 10- to 60-fold increase over amounts previously reported. Autoradiographs of solubilized, electrophoresed membranes show about 30 discrete labeled polypeptides which remain associated with the thylakoid membranes.     

The internal ribosome entry site-mediated translation of antiapoptotic protein XIAP is modulated by the heterogeneous nuclear ribonucleoproteins C1 and C2

The X-chromosome-linked inhibitor of apoptosis, XIAP, is the most powerful and ubiquitous intrinsic inhibitor of apoptosis. We have shown previously that the translation of XIAP is controlled by a potent internal ribosome entry site (IRES) element. IRES-mediated translation of XIAP is increased in response to cellular stress, suggesting the critical role for IRES translation during cellular stress. Here, we demonstrate that heterogeneous nuclear ribonucleoproteins C1 and C2 (hnRNPC1 and -C2) are part of the RNP complex that forms on XIAP IRES. Furthermore, the cellular levels of hnRNPC1 and -C2 parallel the activity of XIAP IRES and the overexpression of hnRNPC1 and -C2 specifically enhanced translation of XIAP IRES, suggesting that hnRNPC1 and -C2 may modulate XIAP expression. Given the central role of XIAP in the regulation of apoptosis these results are important for our understanding of the control of apoptosis.     

Protein building blocks preserved by recombination

Borrowing concepts from the schema theory of genetic algorithms, we have developed a computational algorithm to identify the fragments of proteins, or schemas, that can be recombined without disturbing the integrity of the three-dimensional structure. When recombination leaves these schemas undisturbed, the hybrid proteins are more likely to be folded and functional. Crossovers found by screening libraries of several randomly shuffled proteins for functional hybrids strongly correlate with those predicted by this approach. Experimental results from the construction of hybrids of two beta-lactamases that share 40% amino acid identity demonstrate a threshold in the amount of schema disruption that the hybrid protein can tolerate. To the extent that introns function to promote recombination within proteins, natural selection would serve to bias their locations to schema boundaries.     

Structural aspects of translation termination on the ribosome

Translation of genetic information encoded in messenger RNAs into polypeptide sequences is carried out by ribosomes in all organisms. When a full protein is synthesized, a stop codon positioned in the ribosomal A site signals termination of translation and protein release. Translation termination depends on class I release factors. Recently, atomic-resolution crystal structures were determined for bacterial 70S ribosome termination complexes bound with release factors RF1 or RF2. In combination with recent biochemical studies, the structures resolve long-standing questions about translation termination. They bring insights into the mechanisms of recognition of all three stop codons, peptidyl-tRNA hydrolysis, and coordination of stop-codon recognition with peptidyl-tRNA hydrolysis. In this review, the structural aspects of these mechanisms are discussed.     

Engaging the ribosome: universal IFs of translation

Eukaryotic initiation factor 1A (eIF1A) and the GTPase IF2/eIF5B are the only universally conserved translation initiation factors. Recent structural, biochemical and genetic data indicate that these two factors form an evolutionarily conserved structural and functional unit in translation initiation. Based on insights gathered from studies of the translation elongation factor GTPases, we propose that these factors occupy the aminoacyl-tRNA site (A site) on the ribosome, and promote initiator tRNA binding and ribosomal subunit joining. These processes yield a translationally competent ribosome with Met-tRNA in the ribosomal peptidyl-tRNA site (P site), base-paired to the AUG start codon of a mRNA.     

Evidence that free polysomes are not the precursors of membrane-bound polysomes in rat liver

We tested, in rat liver, the postulate that free polysomes were precursors of membrane-bound polysomes. Three methods were used to isolate free and membrane-bound ribosomes from either post-nuclear or post-mitochondrial supernatants of rat liver. Isolation and quantitation of 28 S and 18 S rRNA allowed determination of the 40 S and 60 S subunit composition of free and membrane-bound ribosomal populations, while pulse labeling of 28 S and 18 S rRNA with [6-¹⁴C]orotic acid and inorganic [³²P]phosphate allowed assessment of relative rates of subunit renewal. Throughout the extra-nuclear compartment, 40 S and 60 S subunits were present in essentially equal numbers, but, free ribosomes contained a stoichiometric excess of 40 S subunits, while membrane-bound ribosomes contained a complementary excess of 60 S subunits. Experiments with labeled precursors showed that throughout the extra-nuclear compartment, 40 S and 60 S subunits accumulated isotopes at essentially equal rates, however, free ribosomes accumulated isotopes faster than membrane-bound ribosomes. Among free ribosomes or polysomes, 40 S subunits accumulated isotopes faster than 60 S subunits, but, this relationship was not seen among membrane-bound ribosomes. Here, 40 S subunits accumulated isotope more slowly than 60 S subunits. This distribution of labeled precursors does not support the postulate that free polysomes are precursors of membrane-bound polysomes, but, these data suggest that membrane-bound polysomes could be precursors of free polysomes.     

Mouse p56 blocks a distinct function of eukaryotic initiation factor 3 in translation initiation

Members of the p56 family of mammalian proteins are strongly induced in virus-infected cells and in cells treated with interferons or double-stranded RNA. Previously, we have reported that human p56 inhibits initiation of translation by binding to the "e" subunit of eukaryotic initiation factor 3 (eIF3) and subsequently interfering with the eIF3/eIF2.GTP.Met-tRNAi (ternary complex) interaction. Here we report that mouse p56 also interferes with eIF3 functions and inhibits translation. However, the murine protein binds to the "c" subunit, not the "e" subunit, of eIF3. Consequently, it has only a marginal effect on eIF3.ternary complex interaction. Instead, the major inhibitory effect of mouse p56 is manifested at a different step of translation initiation, namely the binding of eIF4F to the 40 S ribosomal subunit.eIF3.ternary complex. Thus, mouse and human p56 proteins block different functions of eIF3 by binding to its different subunits.     

Granule neurons in cerebellum express distinct splice variants of the inositol trisphosphate receptor that are modulated by calcium

Primary cultures of granule cells (GC) from rat cerebellar cortex were used to determine whether bioelectric activity, via a Ca²⁺/calmodulin-dependent kinase (CaMK) signaling cascade, modulates expression and exon selection in the inositol trisphosphate receptor type 1 (IP₃R1). IP₃R1 contains or lacks three exons (S1, S2, and S3) that are regulated in a regionally and temporally specific manner. The neuronal, or long, form of IP₃R1 is distinguished from peripheral tissues by inclusion of the S2 exon. Although previous studies indicated that IP₃R1 are undetectable in the cerebellar granular layer in vivo, receptor protein and mRNA are induced in cultured GC grown in medium supplemented with 25 mM KCl or NMDA, two trophic agents that promote long-term survival, compared with GC grown in 5 mM KCl. IP₃R1 induction in response to 25 mM KCl or NMDA is attenuated by coaddition of voltage-sensitive calcium channel or NMDA receptor antagonists, respectively. Actinomycin D, CaMK, and calcineurin antagonists likewise suppress induction. Unlike the major variants of IP₃R1 in Purkinje neurons, which lack S1 and S3, GC grown with trophic agents express mRNA containing these exons. Both neuronal types contain S2. Evidence obtained using mutant mice with Purkinje cell lesions, laser-microdissected GC neurons from slices, and explant cultures indicates that GC predominantly express the S1-containing variant of IP₃R1 in vivo. calmodulin; exon selection     

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.     

Dissecting eukaryotic translation and its control by ribosome density mapping

Translation of an mRNA is generally divided into three stages: initiation, elongation and termination. The relative rates of these steps determine both the number and position of ribosomes along the mRNA, but traditional velocity sedimentation assays for the translational status of mRNA determine only the number of bound ribosomes. We developed a procedure, termed Ribosome Density Mapping (RDM), that uses site-specific cleavage of polysomal mRNA followed by separation on a sucrose gradient and northern analysis, to determine the number of ribosomes associated with specified portions of a particular mRNA. This procedure allows us to test models for translation and its control, and to examine properties of individual steps of translation in vivo. We tested specific predictions from the current model for translational control of GCN4 expression in yeast and found that ribosomes were differentially associated with the uORFs elements and coding region under different growth conditions, consistent with this model. We also mapped ribosome density along the ORF of several mRNAs, to probe basic kinetic properties of translational steps in yeast. We found no detectable decline in ribosome density between the 5′ and 3′ ends of the ORFs, suggesting that the average processivity of elongation is very high. Conversely, there was no queue of ribosomes at the termination site, suggesting that termination is not very slow relative to elongation and initiation. Finally, the RDM results suggest that less frequent initiation of translation on mRNAs with longer ORFs is responsible for the inverse correlation between ORF length and ribosomal density that we observed in a global analysis of translation. These results provide new insights into eukaryotic translation in vivo.     

Model of ribosome translation and mRNA unwinding

A ribosome is an enzyme that catalyzes translation of the genetic information encoded in messenger RNA (mRNA) into proteins. Besides translation through the single-stranded mRNA, the ribosome is also able to translate through the duplex region of mRNA via unwinding the duplex. Here, based on our proposed ribosome translation model, we study analytically the dynamics of Escherichia coli ribosome translation through the duplex region of mRNA, and compare with the available single molecule experimental data. It is shown that the ribosome uses only one active mechanism (mechanical unwinding), rather than two active mechanisms (open-state stabilization and mechanical unwinding), as proposed before, to unwind the duplex. The reduced rate of translation through the duplex region is due to the occurrence of futile transitions, which are induced by the energy barrier from the duplex unwinding to the forward translocation along the single-stranded mRNA. Moreover, we also present predicted results of the average translation rate versus the external force acting on the ribosome translating through the duplex region and through the single-stranded region of mRNA, which can be easily tested by future experiments.