Nanosecond Dynamics of Calmodulin and Ribosome‐Bound Nascent Chains Studied by Time‐Resolved Fluorescence Anisotropy

We report a time‐resolved fluorescence anisotropy study of ribosome‐bound nascent chains (RNCs) of calmodulin (CaM), a prototypical member of the EF‐hand family of calcium‐sensing proteins. As shown in numerous studies, in vitro protein refolding can differ substantially from biosynthetic protein folding, which takes place cotranslationally and depends on the rate of polypeptide chain elongation. A challenge in this respect is to characterize the adopted conformations of nascent chains before their release from the ribosome. CaM RNCs (full‐length, half‐length, and first EF‐hand only) were synthesized in vitro. All constructs contained a tetracysteine motif site‐specifically incorporated in the first N‐terminal helix; this motif is known to react with FlAsH, a biarsenic fluorescein derivative. As the dye is rotationally locked to this helix, we characterized the structural properties and folding states of polypeptide chains tethered to ribosomes and compared these with released chains. Importantly, we observed decelerated tumbling motions of ribosome‐tethered and partially folded nascent chains, compared to released chains. This indicates a pronounced interaction between nascent chains and the ribosome surface, and might reflect chaperone activity of the ribosome.     

Current Views of the Structure of the Mammalian Mitochondrial Ribosome

Mammalian mitochondria synthesize polypeptides crucial for energy generation using ribosomes with a number of unique features. These ribosomes are very protein rich and have very truncated ribosomal RNAs. The bulk of the mammalian mitochondrial ribosome is composed of proteins, only about half of which are homologs of ribosomal proteins found in other translational systems. A number of distinctive features are found in these ribosomes. Among these is a gate-like structure that allows entrance of the primarily leaderless mRNAs that characterize this system. The exit tunnel of the large subunit is also quite unusual and includes a site in which the nascent peptide is visible to solvent prior to the normal exit site. Further, this region of the mitochondrial ribosome is dominated by ribosomal proteins rather than rRNA and is involved in the interaction of the ribosome with the inner membrane where all of the translation products are ultimately located. The proteins of the mitochondrial ribosome appear to play a number of important roles in the cell in addition to their function in protein biosynthesis, including roles in apoptosis and in cell cycle control.     

Ring-opening polymerization of cyclic esters by cyclodextrins

Synthetic polymers, typically prepared by addition polymerization or stepwise polymerization, are used constantly in our daily lives. In recent years, polymer scientists have focused on more environmentally friendly synthetic methods such as mild reaction conditions and biodegradable condensation polymers, including polyesters and polyamides. However, challenges remain in finding greener methods for the synthesis of polymers. Although reactions carried out in water are more environmentally friendly than those in organic solvents, aqueous media can lead to the hydrolysis of condensation polymers. Furthermore, bulk polymerizations are difficult to control. In biological systems, enzymes synthesize most polymers (proteins, DNAs, RNAs, and polysaccharides) in aqueous environments or in condensed phases (membranes). Most enzymes, such as DNA polymerases, RNA polymerases, and ribosomes, form doughnutlike shapes, which encircle the growing polymer chain. As biopolymers form, the active sites and the substrate-combining sites are located at the end of the growing polymer chain and carefully control the polymerization. Therefore, a synthetic catalyst that could insert the monomers between the active site and binding site would create an ideal biomimetic polymerization system. In this Account, we describe cyclodextrins (CDs) as catalysts that can polymerize cyclic esters (lactones and lactides). CDs can initiate polymerizations of cyclic esters in bulk without solvents (even water) to give products in high yields. During our studies on the polymerization of lactones by CDs in bulk, we found that CDs function not only as initiators (catalysts) but also as supporting architectures similar to chaperone proteins. CDs encircle a linear polymer chain so that the chain assumes the proper conformation and avoids coagulation. The CDs can mimic the strategy that living systems use to prepare polymers. Thus, we can obtain polyesters tethered to CDs without employing additional solvents or cocatalysts. Although CD has many hydroxyl groups, only one secondary hydroxyl group attaches to the polyester chain. In addition, the polymerization is highly specific for monomer substrates. We believe that this is the first system in which the catalyst includes monomers initially and subsequently activates the included monomers. The catalyst then inserts the monomers between the binding site and the growing chain. Therefore, this system should provide a new environmentally friendly route to produce biodegradable functional polymers.     

Getting ready to translate: cytoplasmic maturation of eukaryotic ribosomes

The ribosome is the 'universal ribozyme' that is responsible for the final step of decoding genetic information into proteins. While the function of the ribosome is being elucidated at the atomic level, in comparison, little is known regarding its assembly in vivo and intracellular transport. In contrast to prokaryotic ribosomes, the construction of eukaryotic ribosomes, which begins in the nucleolus, requires >200 evolutionary conserved non-ribosomal trans-acting factors, which transiently associate with pre-ribosomal subunits at distinct assembly stages and perform specific maturation steps. Notably, pre-ribosomal subunits are transported to the cytoplasm in a functionally inactive state where they undergo maturation prior to entering translation. In this review, I will summarize our current knowledge of the eukaryotic ribosome assembly pathway with emphasis on cytoplasmic maturation events that render pre-ribosomal subunits translation competent.     

Post‐polymerization modification of bio‐based polymers: maximizing the high functionality of polymers derived from biomass

The renaissance of the bio‐based chemical industry over the last 20 years has seen an ever growing interest in the synthesis of new bio‐based polymers. The building blocks of these new polymers, so called platform molecules, contain significantly more chemical functionality than their petrochemical counterparts (such as ethene, propene and para‐xylene). As a result bio‐based polymers often contain greater residual chemical functionality in their chains, with groups such as alkenes and hydroxyls commonly observed. These functional groups can act as sites for post‐polymerization modification (PPM), thus further extending the range of applications for bio‐based polymers by tailoring the polymers' final properties. This mini‐review highlights some of the most recent and compelling examples of how to make use of bio‐based polymers with residual functional groups for PPM. It also looks at how the emerging interdisciplinary field of enzymatic polymer synthesis allows for increased functionality in polymers by avoiding side‐reactions as a result of milder reaction conditions, and additionally offers an alternative means of polymer surface modification. © 2018 Society of Chemical Industry     

Saccharomyces cerevisiae,a Powerful Model for Studying rRNA Modifications and Their Effects on Translation Fidelity

Ribosomal RNA is a major component of the ribosome. This RNA plays a crucial role in ribosome functioning by ensuring the formation of the peptide bond between amino acids and the accurate decoding of the genetic code. The rRNA carries many chemical modifications that participate in its maturation, the formation of the ribosome and its functioning. In this review, we present the different modifications and how they are deposited on the rRNA. We also describe the most recent results showing that the modified positions are not 100% modified, which creates a heterogeneous population of ribosomes. This gave rise to the concept of specialized ribosomes that we discuss. The knowledge accumulated in the yeast Saccharomyces cerevisiae is very helpful to better understand the role of rRNA modifications in humans, especially in ribosomopathies.     

Abnormal Expression of Mitochondrial Ribosomal Proteins and Their Encoding Genes with Cell Apoptosis and Diseases

Mammalian mitochondrial ribosomes translate 13 proteins encoded by mitochondrial genes, all of which play roles in the mitochondrial respiratory chain. After a long period of reconstruction, mitochondrial ribosomes are the most protein-rich ribosomes. Mitochondrial ribosomal proteins (MRPs) are encoded by nuclear genes, synthesized in the cytoplasm and then, transported to the mitochondria to be assembled into mitochondrial ribosomes. MRPs not only play a role in mitochondrial oxidative phosphorylation (OXPHOS). Moreover, they participate in the regulation of cell state as apoptosis inducing factors. Abnormal expressions of MRPs will lead to mitochondrial metabolism disorder, cell dysfunction, etc. Many researches have demonstrated the abnormal expression of MRPs in various tumors. This paper reviews the basic structure of mitochondrial ribosome, focuses on the structure and function of MRPs, and their relationships with cell apoptosis and diseases. It provides a reference for the study of the function of MRPs and the disease diagnosis and treatment.     

Cover Feature: Molecular Highway Patrol for Ribosome Collisions (ChemBioChem 20/2023)

During translation, messenger RNAs (mRNAs) are decoded by ribosomes which can stall for various reasons. These include chemical damage, codon composition, starvation, or translation inhibition. Trailing ribosomes can collide with stalled ribosomes, potentially leading to dysfunctional or toxic proteins. Such aberrant proteins can form aggregates and favor diseases, especially neurodegeneration. To prevent this, both eukaryotes and bacteria have evolved different pathways to remove faulty nascent peptides, mRNAs and defective ribosomes from the collided complex. In eukaryotes, ubiquitin ligases play central roles in triggering downstream responses and several complexes have been characterized that split affected ribosomes and facilitate degradation of the various components. As collided ribosomes signal translation stress to affected cells, in eukaryotes additional stress response pathways are triggered when collisions are sensed. These pathways inhibit translation and modulate cell survival and immune responses. Here, we summarize the current state of knowledge about rescue and stress response pathways triggered by ribosome collisions.     

A Recombinant Collagen–mRNA Platform for Controllable Protein Synthesis

We have developed a collagen–mRNA platform for controllable protein production that is intended to be less prone to the problems associated with commonly used mRNA therapy as well as with collagen skin‐healing procedures. A collagen mimic was constructed according to a recombinant method and was used as scaffold for translating mRNA chains into proteins. Cysteines were genetically inserted into the collagen chain at positions allowing efficient ribosome translation activity while minimizing mRNA misfolding and degradation. Enhanced green fluorescence protein (eGFP) mRNA bound to collagen was successfully translated by cell‐free Escherichia coli ribosomes. This system enabled an accurate control of specific protein synthesis by monitoring expression time and level. Luciferase–mRNA was also translated on collagen scaffold by eukaryotic cell extracts. Thus we have demonstrated the feasibility of controllable protein synthesis on collagen scaffolds by ribosomal machinery.     

Helix 69: A Multitasking RNA Motif as a Novel Drug Target

High-resolution structures have shown that helix 69 (H69) of the large ribosomal subunit can assume variable conformational states during translation. Solution studies on small model RNAs, isolated subunits, and complete ribosomes also revealed a variety of H69 conformations. Specific nucleotides of H69 that undergo changes in their relative orientations within the ribosome structure play important roles in both translation and ribosome rescue. Furthermore, the presence of multiple pseudouridines influences the global conformational states of H69, and these highly conserved modifications play a role in regulating the positioning of key functional residues. Helix 69 has recently been identified as a novel antibiotic target site. Small molecules such as aminoglycosides bind to specific conformational states of H69 in bacterial ribosomes and affect the translation process. A variety of techniques have been employed to study H69, ranging from chemical synthesis to X-ray crystallography, highlighting the importance of a detailed evaluation of the underlying principles of RNA conformational dynamics and drug targeting.     

The Proto-Ribosome: An Ancient Nano-machine for Peptide Bond Formation

The ribosome is a ribozyme whose active site, the peptidyl transferase center (PTC), is situated within a highly conserved universal symmetrical region that connects all ribosomal functional centers involved in amino acid polymerization. The linkage between this elaborate architecture and A-site tRNA position revealed that the A- to P-site passage of the tRNA 3′ terminus during protein synthesis is performed by a rotary motion, synchronized with the overall tRNA/mRNA sideways movement, and guided by the PTC. This rotary motion leads to suitable stereochemistry for peptide bond formation as well as for substrate-mediated catalysis. Analysis of the substrate binding modes to ribosomes led to the hypothesis that the ancient ribosome produced single peptide bonds and non-coded chains, potentially in a similar manner to the modern PTC. Later in evolution, a mechanism, enabling some type of decoding genetic control triggered the emergence of the small ribosomal subunit or part of it. This seems to be the result of the appearance of reaction products that could have evolved after polypeptides capable of enzymatic function were generated sporadically, while an ancient stable RNA fold was converted into an old version of a tRNA molecule. Since in the contemporary ribosome, the symmetry relates only to the backbone fold and nucleotide orientations but not nucleotide sequences, it emphasizes the superiority of functional requirement over sequence conservation, and indicates that the PTC may have evolved by gene fusion or gene duplication.     

Ribosome: The Structure–Function Relation and a New Paradigm to the Protein Folding Problem

The rate of protein synthesis is about seven and fifteen amino acids per second, in the eukaryotic and the bacterial ribosome, respectively. Hence, a few minutes is required to synthesize a polypeptide of an average length. This is much longer than the time needed for the hydrophobic collapse (folding) to take place. So a polypeptide gets enough time to form its local secondary to tertiary structures cotranslationally and put such segments in proper order while in association with the ribosome, unless something prevents its entire length from folding. As reported earlier, ribosomes from prokaryotes, eukaryotes, and mitochondria act as molds for protein folding, and each mold has a set of recognition sites for all proteins. More specifically, the mold is the peptidyl transferase center (PTC), a part of the large RNA of the large ribosomal subunit. Specific amino acids from different random coil regions in a protein interact with specific nucleotides in the PTC, which brings the entire length of the protein into the small space of the PTC mold. The mold thus helps to stabilize the entropy-driven collapsed state of the polypeptide. The process also divides the protein into small segments; each segment is connected at two ends with two nucleotides and can fold in the ribosomal environment. The segments dissociate in such a sequence that the organization proceeds hierarchically from the core of the globular protein radially towards the outer surface. Then the protein dissociates from the ribosome in a “folding competent state” which does the final fine tuning in folding outside the ribosome. While the ribosomal contact and release are over in 1–2 minutes in vitro, the fine tuning takes about 5–10 minutes. Release from the ribosome needs no added energy factor from outside, like ATP.     

Ribosome Biogenesis and Cancer: Overview on Ribosomal Proteins

Cytosolic ribosomes (cytoribosomes) are macromolecular ribonucleoprotein complexes that are assembled from ribosomal RNA and ribosomal proteins, which are essential for protein biosynthesis. Mitochondrial ribosomes (mitoribosomes) perform translation of the proteins essential for the oxidative phosphorylation system. The biogenesis of cytoribosomes and mitoribosomes includes ribosomal RNA processing, modification and binding to ribosomal proteins and is assisted by numerous biogenesis factors. This is a major energy-consuming process in the cell and, therefore, is highly coordinated and sensitive to several cellular stressors. In mitochondria, the regulation of mitoribosome biogenesis is essential for cellular respiration, a process linked to cell growth and proliferation. This review briefly overviews the key stages of cytosolic and mitochondrial ribosome biogenesis; summarizes the main steps of ribosome biogenesis alterations occurring during tumorigenesis, highlighting the changes in the expression level of cytosolic ribosomal proteins (CRPs) and mitochondrial ribosomal proteins (MRPs) in different types of tumors; focuses on the currently available information regarding the extra-ribosomal functions of CRPs and MRPs correlated to cancer; and discusses the role of CRPs and MRPs as biomarkers and/or molecular targets in cancer treatment.     

Fatty acid‐based comonomers as styrene replacements in soybean and castor oil‐based thermosetting polymers

In this study, a fatty acid‐based comonomer is employed as a styrene replacement for the production of triglyceride‐based thermosetting resins. Styrene is a hazardous pollutant and a volatile organic compound. Given their low volatility, fatty acid monomers, such as methacrylated lauric acid (MLA), are attractive alternatives in reducing or eliminating styrene usage. Different triglyceride‐derived cross‐linkers resins were produced for this purpose: acrylated epoxidized soybean oil (AESO), maleinated AESO (MAESO), maleinated soybean oil monoglyceride (SOMG/MA) and maleinated castor oil monoglyceride (COMG/MA). The mechanical properties of the bio‐based polymers and the viscosities of bio‐based resins were analyzed. The viscosities of the resins using MLA were higher than that of resins with styrene. Decreasing the content of MLA increased the glass transition temperature (T_g). In fact, the T_g of bio‐based resin/MLA polymers were on the order of 60°C, which was significantly lower than the bio‐based resin/styrene polymers. Ternary blends of SOMG/MA and COMG/MA with MLA and styrene improved the mechanical properties and reduced the resin viscosity to acceptable values. Lastly, butyrated kraft lignin was incorporated into the bio‐based resins, ultimately leading to improved mechanical properties of this thermoset but with unacceptable increases in viscosity. © 2010 Wiley Periodicals, Inc. J Appl Polym Sci, 2011     

Progress in the Preparation of Functional and (Bio)Degradable Polymers via Living Polymerizations

This review presents the latest developments in (bio)degradable approaches and functional aliphatic polyesters and polycarbonates prepared by typical ring-opening polymerization (ROP) of lactones and trimethylene carbonates. It also considers several recent innovative synthetic methods including radical ring-opening polymerization (RROP), atom transfer radical polyaddition (ATRPA), and simultaneous chain- and step-growth radical polymerization (SCSRP) that produce aliphatic polyesters. With regard to (bio)degradable approaches, we have summarized several representative cleavable linkages that make it possible to obtain cleavable polymers. In the section on functional aliphatic polyesters, we explore the syntheses of specific functional lactones, which can be performed by ring-opening copolymerization of typical lactone/lactide monomers. Last but not the least, in the recent innovative methods section, three interesting synthetic methodologies, RROP, ATRPA, and SCSRP are discussed in detail with regard to their reaction mechanisms and polymer functionalities.     

tRNA Modification Enzymes GidA and MnmE: Potential Role in Virulence of Bacterial Pathogens

Transfer RNA (tRNA) is an RNA molecule that carries amino acids to the ribosomes for protein synthesis. These tRNAs function at the peptidyl (P) and aminoacyl (A) binding sites of the ribosome during translation, with each codon being recognized by a specific tRNA. Due to this specificity, tRNA modification is essential for translational efficiency. Many enzymes have been implicated in the modification of bacterial tRNAs, and these enzymes may complex with one another or interact individually with the tRNA. Approximately, 100 tRNA modification enzymes have been identified with glucose-inhibited division (GidA) protein and MnmE being two of the enzymes studied. In Escherichia coli and Salmonella, GidA and MnmE bind together to form a functional complex responsible for the proper biosynthesis of 5-methylaminomethyl-2-thiouridine (mnm⁵s²U34) of tRNAs. Studies have implicated this pathway in a major pathogenic regulatory mechanism as deletion of gidA and/or mnmE has attenuated several bacterial pathogens like Salmonella enterica serovar Typhimurium, Pseudomonas syringae, Aeromonas hydrophila, and many others. In this review, we summarize the potential role of the GidA/MnmE tRNA modification pathway in bacterial virulence, interactions with the host, and potential therapeutic strategies resulting from a greater understanding of this regulatory mechanism.