Cell-free biosynthesis and engineering of ribosomally synthesized lanthipeptides.

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a major class of natural products with diverse chemical structures and potent biological activities. A vast majority of RiPP gene clusters remain unexplored in microbial genomes, which is partially due to the lack of rapid and efficient heterologous expression systems for RiPP characterization and biosynthesis. Here, we report a unified biocatalysis (UniBioCat) system based on cell-free gene expression for rapid biosynthesis and engineering of RiPPs. We demonstrate UniBioCat by reconstituting a full biosynthetic pathway for de novo biosynthesis of salivaricin B, a lanthipeptide RiPP. Next, we delete several protease/peptidase genes from the source strain to enhance the performance of UniBioCat, which then can synthesize and screen salivaricin B variants with enhanced antimicrobial activity. Finally, we show that UniBioCat is generalizable by synthesizing and evaluating the bioactivity of ten uncharacterized lanthipeptides. We expect UniBioCat to accelerate the discovery, characterization, and synthesis of RiPPs.

5'UTR G-quadruplex structure enhances translation in size dependent manner.

Translation initiation in bacteria is frequently regulated by various structures in the 5' untranslated region (5'UTR). Previously, we demonstrated that G-quadruplex (G4) formation in non-template DNA enhances transcription. In this study, we aim to explore how G4 formation in mRNA (RG4) at 5'UTR impacts translation using a T7-based in vitro translation system and in E. coli. We show that RG4 strongly promotes translation efficiency in a size-dependent manner. Additionally, inserting a hairpin upstream of the RG4 further enhances translation efficiency, reaching up to a 12-fold increase. We find that the RG4-dependent effect is not due to increased ribosome affinity, ribosome binding site accessibility, or mRNA stability. We propose a physical barrier model in which bulky structures in 5'UTR biases ribosome movement toward the downstream start codon, thereby increasing the translation output. This study provides biophysical insights into the regulatory role of 5'UTR structures in in vitro and bacterial translation, highlighting their potential applications in tuning gene expression.

Microorganisms in subarctic soils are depleted of ribosomes under short-, medium-, and long-term warming.

Physiological responses of soil microorganisms to global warming are important for soil ecosystem function and the terrestrial carbon cycle. Here, we investigate the effects of weeks, years, and decades of soil warming across seasons and time on the microbial protein biosynthesis machineries (i.e. ribosomes), the most abundant cellular macromolecular complexes, using RNA:DNA and RNA:MBC (microbial biomass carbon) ratios as proxies for cellular ribosome contents. We compared warmed soils and non-warmed controls of 15 replicated subarctic grassland and forest soil temperature gradients subject to natural geothermal warming. RNA:DNA ratios tended to be lower in the warmed soils during summer and autumn, independent of warming duration (6 weeks, 8-14 years, and > 50 years), warming intensity (+3°C, +6°C, and +9°C), and ecosystem type. With increasing temperatures, RNA:MBC ratios were also decreasing. Additionally, seasonal RNA:DNA ratios of the consecutively sampled forest showed the same temperature-driven pattern. This suggests that subarctic soil microorganisms are depleted of ribosomes under warm conditions and the lack of consistent relationships with other physicochemical parameters besides temperature further suggests temperature as key driver. Furthermore, in incubation experiments, we measured significantly higher CO2 emission rates per unit of RNA from short- and long-term warmed soils compared to non-warmed controls. In conclusion, ribosome reduction may represent a widespread microbial physiological response to warming that offers a selective advantage at higher temperatures, as energy and matter can be reallocated from ribosome synthesis to other processes including substrate uptake and turnover. This way, ribosome reduction could have a substantial effect on soil carbon dynamics.

Decoding the ribosome's hidden language: rRNA modifications as key players in cancer dynamics and targeted therapies.

Ribosomal RNA (rRNA) modifications, essential components of ribosome structure and function, significantly impact cellular proteomics and cancer biology. These chemical modifications transcend structural roles, critically shaping ribosome functionality and influencing cellular protein profiles. In this review, the mechanisms by which rRNA modifications regulate both rRNA functions and broader cellular physiological processes are critically discussed. Importantly, by altering the translational output, rRNA modifications can shift the cellular equilibrium towards oncogenesis, thus playing a key role in cancer development and progression. Moreover, a special focus is placed on the functions of mitochondrial rRNA modifications and their aberrant expression in cancer, an area with profound implications yet largely uncharted. Dysregulation in these modifications can lead to metabolic dysfunction and apoptosis resistance, hallmark traits of cancer cells. Furthermore, the current challenges and future perspectives in targeting rRNA modifications are highlighted as a therapeutic approach for cancer treatment. In conclusion, rRNA modifications represent a frontier in cancer research, offering novel insights and therapeutic possibilities. Understanding and harnessing these modifications can pave the way for breakthroughs in cancer treatment, potentially transforming the approach to combating this complex disease.

The Structural and Molecular Mechanisms of Mycobacterium tuberculosis Translational Elongation Factor Proteins.

Targeting translation factor proteins holds promise for developing innovative anti-tuberculosis drugs. During protein translation, many factors cause ribosomes to stall at messenger RNA (mRNA). To maintain protein homeostasis, bacteria have evolved various ribosome rescue mechanisms, including the predominant trans-translation process, to release stalled ribosomes and remove aberrant mRNAs. The rescue systems require the participation of translation elongation factor proteins (EFs) and are essential for bacterial physiology and reproduction. However, they disappear during eukaryotic evolution, which makes the essential proteins and translation elongation factors promising antimicrobial drug targets. Here, we review the structural and molecular mechanisms of the translation elongation factors EF-Tu, EF-Ts, and EF-G, which play essential roles in the normal translation and ribosome rescue mechanisms of Mycobacterium tuberculosis (Mtb). We also briefly describe the structure-based, computer-assisted study of anti-tuberculosis drugs.

High-confidence 3D template matching for cryo-electron tomography.

Visual proteomics attempts to build atlases of the molecular content of cells but the automated annotation of cryo electron tomograms remains challenging. Template matching (TM) and methods based on machine learning detect structural signatures of macromolecules. However, their applicability remains limited in terms of both the abundance and size of the molecular targets. Here we show that the performance of TM is greatly improved by using template-specific search parameter optimization and by including higher-resolution information. We establish a TM pipeline with systematically tuned parameters for the automated, objective and comprehensive identification of structures with confidence 10 to 100-fold above the noise level. We demonstrate high-fidelity and high-confidence localizations of nuclear pore complexes, vaults, ribosomes, proteasomes, fatty acid synthases, lipid membranes and microtubules, and individual subunits inside crowded eukaryotic cells. We provide software tools for the generic implementation of our method that is broadly applicable towards realizing visual proteomics.

Reflections on the Origin of Coded Protein Biosynthesis.

The principle of continuity posits that some central features of primordial biocatalytic mechanisms should still be present in the genetically dependent pathway of protein synthesis, a crucial step in the emergence of life. Key bimolecular reactions of this process are catalyzed by DNA-dependent RNA polymerases, aminoacyl-tRNA synthetases, and ribosomes. Remarkably, none of these biocatalysts contribute chemically active groups to their respective reactions. Instead, structural and functional studies have demonstrated that nucleotidic α-phosphate and ß-d-ribosyl 2' OH and 3' OH groups can help their own catalysis, a process which, consequently, has been called "substrate-assisted". Furthermore, upon binding, the substrates significantly lower the entropy of activation, exclude water from these catalysts' active sites, and are readily positioned for a reaction. This binding mode has been described as an "entropy trap". The combination of this effect with substrate-assisted catalysis results in reactions that are stereochemically and mechanistically simpler than the ones found in most modern enzymes. This observation is consistent with the way in which primordial catalysts could have operated; it may also explain why, thanks to their complementary reactivities, ß-d-ribose and phosphate were naturally selected to be the central components of early coding polymers.

Exploring the roles of ribosomal peptides in prokaryote-phage interactions through deep learning-enabled metagenome mining.

BACKGROUND: Microbial secondary metabolites play a crucial role in the intricate interactions within the natural environment. Among these metabolites, ribosomally synthesized and post-translationally modified peptides (RiPPs) are becoming a promising source of therapeutic agents due to their structural diversity and functional versatility. However, their biosynthetic capacity and ecological functions remain largely underexplored. RESULTS: Here, we aim to explore the biosynthetic profile of RiPPs and their potential roles in the interactions between microbes and viruses in the ocean, which encompasses a vast diversity of unique biomes that are rich in interactions and remains chemically underexplored. We first developed TrRiPP to identify RiPPs from ocean metagenomes, a deep learning method that detects RiPP precursors in a hallmark gene-independent manner to overcome the limitations of classic methods in processing highly fragmented metagenomic data. Applying this method to metagenomes from the global ocean microbiome, we uncover a diverse array of previously uncharacterized putative RiPP families with great novelty and diversity. Through correlation analysis based on metatranscriptomic data, we observed a high prevalence of antiphage defense-related and phage-related protein families that were co-expressed with RiPP families. Based on this putative association between RiPPs and phage infection, we constructed an Ocean Virus Database (OVD) and established a RiPP-involving host-phage interaction network through host prediction and co-expression analysis, revealing complex connectivities linking RiPP-encoding prokaryotes, RiPP families, viral protein families, and phages. These findings highlight the potential of RiPP families involved in prokaryote-phage interactions and coevolution, providing insights into their ecological functions in the ocean microbiome. CONCLUSIONS: This study provides a systematic investigation of the biosynthetic potential of RiPPs from the ocean microbiome at a global scale, shedding light on the essential insights into the ecological functions of RiPPs in prokaryote-phage interactions through the integration of deep learning approaches, metatranscriptomic data, and host-phage connectivity. This study serves as a valuable example of exploring the ecological functions of bacterial secondary metabolites, particularly their associations with unexplored microbial interactions. Video Abstract.

Loss of Conserved rRNA Modifications in the Peptidyl Transferase Center Leads to Diminished Protein Synthesis and Cell Growth in Budding Yeast.

Ribosomal RNAs (rRNAs) are extensively modified during the transcription and subsequent maturation. Three types of modifications, 2'-O-methylation of ribose moiety, pseudouridylation, and base modifications, are introduced either by a snoRNA-driven mechanism or by stand-alone enzymes. Modified nucleotides are clustered at the functionally important sites, including peptidyl transferase center (PTC). Therefore, it has been hypothesised that the modified nucleotides play an important role in ensuring the functionality of the ribosome. In this study, we demonstrate that seven 25S rRNA modifications, including four evolutionarily conserved modifications, in the proximity of PTC can be simultaneously depleted without loss of cell viability. Yeast mutants lacking three snoRNA genes (snR34, snR52, and snR65) and/or expressing enzymatically inactive variants of spb1(D52A/E679K) and nop2(C424A/C478A) were constructed. The results show that rRNA modifications in PTC contribute collectively to efficient translation in eukaryotic cells. The deficiency of seven modified nucleotides in 25S rRNA resulted in reduced cell growth, cold sensitivity, decreased translation levels, and hyperaccurate translation, as indicated by the reduced missense and nonsense suppression. The modification m5C2870 is crucial in the absence of the other six modified nucleotides. Thus, the pattern of rRNA-modified nucleotides around the PTC is essential for optimal ribosomal translational activity and translational fidelity.

How total mRNA influences cell growth.

Experimental observations tracing back to the 1960s imply that ribosome quantities play a prominent role in determining a cell's growth. Nevertheless, in biologically relevant scenarios, growth can also be influenced by the levels of mRNA and RNA polymerase. Here, we construct a quantitative model of biosynthesis providing testable scenarios for these situations. The model explores a theoretically motivated regime where RNA polymerases compete for genes and ribosomes for transcripts and gives general expressions relating growth rate, mRNA concentrations, ribosome, and RNA polymerase levels. On general grounds, the model predicts how the fraction of ribosomes in the proteome depends on total mRNA concentration and inspects an underexplored regime in which the trade-off between transcript levels and ribosome abundances sets the cellular growth rate. In particular, we show that the model predicts and clarifies three important experimental observations, in budding yeast and Escherichia coli bacteria: i) that the growth-rate cost of unneeded protein expression can be affected by mRNA levels, ii) that resource optimization leads to decreasing trends in mRNA levels at slow growth, and iii) that ribosome allocation may increase, stay constant, or decrease, in response to transcription-inhibiting antibiotics. Since the data indicate that a regime of joint limitation may apply in physiological conditions and not only to perturbations, we speculate that this regime is likely self-imposed.

Multi-omics profiling reveals dysregulated ribosome biogenesis and impaired cell proliferation following knockout of CDR2L.

BACKGROUND: Cerebellar degeneration-related (CDR) proteins are associated with paraneoplastic cerebellar degeneration (PCD) - a rare, neurodegenerative disease caused by tumour-induced autoimmunity against neural antigens resulting in degeneration of Purkinje neurons in the cerebellum. The pathogenesis of PCD is unknown, in large part due to our limited understanding of the functions of CDR proteins. To this end, we performed an extensive, multi-omics analysis of CDR-knockout cells focusing on the CDR2L protein, to gain a deeper understanding of the properties of the CDR proteins in ovarian cancer. METHODS: Ovarian cancer cell lines lacking either CDR1, CDR2, or CDR2L were analysed using RNA sequencing and mass spectrometry-based proteomics to assess changes to the transcriptome, proteome and secretome in the absence of these proteins. RESULTS: For each knockout cell line, we identified sets of differentially expressed genes and proteins. CDR2L-knockout cells displayed a distinct expression profile compared to CDR1- and CDR2-knockout cells. Knockout of CDR2L caused dysregulation of genes involved in ribosome biogenesis, protein translation, and cell cycle progression, ultimately causing impaired cell proliferation in vitro. Several of these genes showed a concurrent upregulation at the transcript level and downregulation at the protein level. CONCLUSIONS: Our study provides the first integrative multi-omics analysis of the impact of knockout of the CDR genes, providing both new insights into the biological properties of the CDR proteins in ovarian cancer, and a valuable resource for future investigations into the CDR proteins.

Ribonuclease inhibitor and angiogenin system regulates cell type-specific global translation.

Translation of mRNAs is a fundamental process that occurs in all cell types of multicellular organisms. Conventionally, it has been considered a default step in gene expression, lacking specific regulation. However, recent studies have documented that certain mRNAs exhibit cell type-specific translation. Despite this, it remains unclear whether global translation is controlled in a cell type-specific manner. By using human cell lines and mouse models, we found that deletion of the ribosome-associated protein ribonuclease inhibitor 1 (RNH1) decreases global translation selectively in hematopoietic-origin cells but not in the non-hematopoietic-origin cells. RNH1-mediated cell type-specific translation is mechanistically linked to angiogenin-induced ribosomal biogenesis. Collectively, this study unravels the existence of cell type-specific global translation regulators and highlights the complex translation regulation in vertebrates.

Nascent chains derived from a foldable protein sequence interact with specific ribosomal surface sites near the exit tunnel.

In order to become bioactive, proteins must be translated and protected from aggregation during biosynthesis. The ribosome and molecular chaperones play a key role in this process. Ribosome-bound nascent chains (RNCs) of intrinsically disordered proteins and RNCs bearing a signal/arrest sequence are known to interact with ribosomal proteins. However, in the case of RNCs bearing foldable protein sequences, not much information is available on these interactions. Here, via a combination of chemical crosslinking and time-resolved fluorescence-anisotropy, we find that nascent chains of the foldable globin apoHmp1-140 interact with ribosomal protein L23 and have a freely-tumbling non-interacting N-terminal compact region comprising 63-94 residues. Longer RNCs (apoHmp1-189) also interact with an additional yet unidentified ribosomal protein, as well as with chaperones. Surprisingly, the apparent strength of RNC/r-protein interactions does not depend on nascent-chain sequence. Overall, foldable nascent chains establish and expand interactions with selected ribosomal proteins and chaperones, as they get longer. These data are significant because they reveal the interplay between independent conformational sampling and nascent-protein interactions with the ribosomal surface.

Lentivirus-mediated gene therapy corrects ribosomal biogenesis and shows promise for Diamond Blackfan anemia.

This study lays the groundwork for future lentivirus-mediated gene therapy in patients with Diamond Blackfan anemia (DBA) caused by mutations in ribosomal protein S19 (RPS19), showing evidence of a new safe and effective therapy. The data show that, unlike patients with Fanconi anemia (FA), the hematopoietic stem cell (HSC) reservoir of patients with DBA was not significantly reduced, suggesting that collection of these cells should not constitute a remarkable restriction for DBA gene therapy. Subsequently, 2 clinically applicable lentiviral vectors were developed. In the former lentiviral vector, PGK.CoRPS19 LV, a codon-optimized version of RPS19 was driven by the phosphoglycerate kinase promoter (PGK) already used in different gene therapy trials, including FA gene therapy. In the latter one, EF1α.CoRPS19 LV, RPS19 expression was driven by the elongation factor alpha short promoter, EF1α(s). Preclinical experiments showed that transduction of DBA patient CD34+ cells with the PGK.CoRPS19 LV restored erythroid differentiation, and demonstrated the long-term repopulating properties of corrected DBA CD34+ cells, providing evidence of improved erythroid maturation. Concomitantly, long-term restoration of ribosomal biogenesis was verified using a potentially novel method applicable to patients' blood cells, based on ribosomal RNA methylation analyses. Finally, in vivo safety studies and proviral insertion site analyses showed that lentivirus-mediated gene therapy was nontoxic.

Translation initiation factor eIF1.2 promotes Toxoplasma stage conversion by regulating levels of key differentiation factors.

The parasite Toxoplasma gondii persists in its hosts by converting from replicating tachyzoites to latent bradyzoites housed in tissue cysts. The molecular mechanisms that mediate T. gondii differentiation remain poorly understood. Through a mutagenesis screen, we identified translation initiation factor eIF1.2 as a critical factor for T. gondii differentiation. A F97L mutation in eIF1.2 or the genetic ablation of eIF1.2 (∆eif1.2) markedly impeded bradyzoite cyst formation in vitro and in vivo. We demonstrated, at single-molecule level, that the eIF1.2 F97L mutation impacts the scanning process of the ribosome preinitiation complex on a model mRNA. RNA sequencing and ribosome profiling experiments unveiled that ∆eif1.2 parasites are defective in upregulating bradyzoite induction factors BFD1 and BFD2 during stress-induced differentiation. Forced expression of BFD1 or BFD2 significantly restored differentiation in ∆eif1.2 parasites. Together, our findings suggest that eIF1.2 functions by regulating the translation of key differentiation factors necessary to establish chronic toxoplasmosis.

TGF-ß2 Induces Ribosome Activity, Alters Ribosome Composition and Inhibits IRES-Mediated Translation in Chondrocytes.

Alterations in cell fate are often attributed to (epigenetic) regulation of gene expression. An emerging paradigm focuses on specialized ribosomes within a cell. However, little evidence exists for the dynamic regulation of ribosome composition and function. Here, we stimulated a chondrocytic cell line with transforming growth factor beta (TGF-ß2) and mapped changes in ribosome function, composition and ribosomal RNA (rRNA) epitranscriptomics. 35S Met/Cys incorporation was used to evaluate ribosome activity. Dual luciferase reporter assays were used to assess ribosomal modus. Ribosomal RNA expression and processing were determined by RT-qPCR, while RiboMethSeq and HydraPsiSeq were used to determine rRNA modification profiles. Label-free protein quantification of total cell lysates, isolated ribosomes and secreted proteins was done by LC-MS/MS. A three-day TGF-ß2 stimulation induced total protein synthesis in SW1353 chondrocytic cells and human articular chondrocytes. Specifically, TGF-ß2 induced cap-mediated protein synthesis, while IRES-mediated translation was not (P53 IRES) or little affected (CrPv IGR and HCV IRES). Three rRNA post-transcriptional modifications (PTMs) were affected by TGF-ß2 stimulation (18S-Gm1447 downregulated, 18S-ψ1177 and 28S-ψ4598 upregulated). Proteomic analysis of isolated ribosomes revealed increased interaction with eIF2 and tRNA ligases and decreased association of eIF4A3 and heterogeneous nuclear ribonucleoprotein (HNRNP)s. In addition, thirteen core ribosomal proteins were more present in ribosomes from TGF-ß2 stimulated cells, albeit with a modest fold change. A prolonged stimulation of chondrocytic cells with TGF-ß2 induced ribosome activity and changed the mode of translation. These functional changes could be coupled to alterations in accessory proteins in the ribosomal proteome.

Multimodal binding and inhibition of bacterial ribosomes by the antimicrobial peptides Api137 and Api88.

Proline-rich antimicrobial peptides (PrAMPs) inhibit bacterial protein biosynthesis by binding to the polypeptide exit tunnel (PET) near the peptidyl transferase center. Api137, an optimized derivative of honeybee PrAMP apidaecin, inhibits protein expression by trapping release factors (RFs), which interact with stop codons on ribosomes to terminate translation. This study uses cryo-EM, functional assays and molecular dynamic (MD) simulations to show that Api137 additionally occupies a second binding site near the exit of the PET and can repress translation independently of RF-trapping. Api88, a C-terminally amidated (-CONH2) analog of Api137 (-COOH), binds to the same sites, occupies a third binding pocket and interferes with the translation process presumably without RF-trapping. In conclusion, apidaecin-derived PrAMPs inhibit bacterial ribosomes by multimodal mechanisms caused by minor structural changes and thus represent a promising pool for drug development efforts.

Exon-junction complex association with stalled ribosomes and slow translation-independent disassembly.

Exon junction complexes are deposited at exon-exon junctions during splicing. They are primarily known to activate non-sense mediated degradation of transcripts harbouring premature stop codons before the last intron. According to a popular model, exon-junction complexes accompany mRNAs to the cytoplasm where the first translating ribosome pushes them out. However, they are also removed by uncharacterized, translation-independent mechanisms. Little is known about kinetic and transcript specificity of these processes. Here we tag core subunits of exon-junction complexes with complementary split nanoluciferase fragments to obtain sensitive and quantitative assays for complex formation. Unexpectedly, exon-junction complexes form large stable mRNPs containing stalled ribosomes. Complex assembly and disassembly rates are determined after an arrest in transcription and/or translation. 85% of newly deposited exon-junction complexes are disassembled by a translation-dependent mechanism. However as this process is much faster than the translation-independent one, only 30% of the exon-junction complexes present in cells at steady state require translation for disassembly. Deep RNA sequencing shows a bias of exon-junction complex bound transcripts towards microtubule and centrosome coding ones and demonstrate that the lifetimes of exon-junction complexes are transcript-specific. This study provides a dynamic vision of exon-junction complexes and uncovers their unexpected stable association with ribosomes.

Integrated ribosome and proteome analyses reveal insights into sevoflurane-induced long-term social behavior and cognitive dysfunctions through ADNP inhibition in neonatal mice.

A growing number of studies have demonstrated that repeated exposure to sevoflurane during development results in persistent social abnormalities and cognitive impairment. Davunetide, an active fragment of the activity-dependent neuroprotective protein (ADNP), has been implicated in social and cognitive protection. However, the potential of davunetide to attenuate social deficits following sevoflurane exposure and the underlying developmental mechanisms remain poorly understood. In this study, ribosome and proteome profiles were analyzed to investigate the molecular basis of sevoflurane-induced social deficits in neonatal mice. The neuropathological basis was also explored using Golgi staining, morphological analysis, western blotting, electrophysiological analysis, and behavioral analysis. Results indicated that ADNP was significantly down-regulated following developmental exposure to sevoflurane. In adulthood, anterior cingulate cortex (ACC) neurons exposed to sevoflurane exhibited a decrease in dendrite number, total dendrite length, and spine density. Furthermore, the expression levels of Homer, PSD95, synaptophysin, and vglut2 were significantly reduced in the sevoflurane group. Patch-clamp recordings indicated reductions in both the frequency and amplitude of miniature excitatory postsynaptic currents (mEPSCs). Notably, davunetide significantly ameliorated the synaptic defects, social behavior deficits, and cognitive impairments induced by sevoflurane. Mechanistic analysis revealed that loss of ADNP led to dysregulation of Ca 2+ activity via the Wnt/ß-catenin signaling, resulting in decreased expression of synaptic proteins. Suppression of Wnt signaling was restored in the davunetide-treated group. Thus, ADNP was identified as a promising therapeutic target for the prevention and treatment of neurodevelopmental toxicity caused by general anesthetics. This study provides important insights into the mechanisms underlying social and cognitive disturbances caused by sevoflurane exposure in neonatal mice and elucidates the regulatory pathways involved.

On the Re-Creation of Protoribosome Analogues in the Lab.

The evolution of the translation system is a fundamental issue in the quest for the origin of life. A feasible evolutionary scenario necessitates the autonomous emergence of a protoribosome capable of catalyzing the synthesis of the initial peptides. The peptidyl transferase center (PTC) region in the modern ribosomal large subunit is believed to retain a vestige of such a prebiotic non-coded protoribosome, which would have self-assembled from random RNA chains, catalyzed peptide bond formation between arbitrary amino acids, and produced short peptides. Recently, three research groups experimentally demonstrated that several distinct dimeric constructs of protoribosome analogues, derived predicated on the approximate 2-fold rotational symmetry inherent in the PTC region, possess the ability to spontaneously fold, dimerize, and catalyze the formation of peptide bonds and of short peptides. These dimers are examined, aiming at retrieving information concerned with the characteristics of a prebiotic protoribosome. The analysis suggests preconditions for the laboratory re-creation of credible protoribosome analogues, including the preference of a heterodimer protoribosome, contradicting the common belief in the precedence of homodimers. Additionally, it derives a dynamic process which possibly played a role in the spontaneous production of the first bio-catalyzed peptides in the prebiotic world.