Michal H. Kolář

Early ribonucleoprotein systems likely required mechanisms to concentrate and organize RNA. Modern ribosomes use Mg ²⁺ and evolved Lys/Arg-rich protein extensions to stabilize their RNA backbone. Before templated synthesis, however, peptide formation likely generated heterogeneous sequence populations rather than reproducible ribosomal sequences. Here, we compare Mg ²⁺ , ribosomal peptides, and statistical peptide libraries in organizing a 136-nucleotide model of the peptidyl transferase center (sPTC). Mg ²⁺ condenses sPTC only at a large excess of positive charge following thermal annealing, producing largely arrested libraries. Acidic conditions further promote condensation. Ribosomal peptides instead promote coacervation near charge stoichiometry and form droplets that readily fuse. Several statistical peptide libraries also coacervate with sPTC, despite comprising heterogenous mixtures rather than a single defined sequence. Increasing mean positive charge favors condensation, but Lys/Arg-containing libraries undergo liquid-liquid phase separation more readily and across broader conditions than matched libraries containing the prebiotically plausible diaminopropionic acid (Dpr) and diaminobutyric acid (Dab). Atomistic computer simulations implicate Arg as a major source of this difference, as it can form more numerous and longer-lived hydrogen bonds with RNA, while competitive partitioning experiments show preferential condensate recruitment in the order Arg > Lys > Dab > Dpr. Together, our findings show that peptide-RNA coacervation can be triggered collectively by statistical peptide ensembles, providing a plausible route to protoribosomal organization without peptide sequence-specific optimization. Cationic chemistry shapes condensate formation and material properties, suggesting that amino acid alphabet formation could have broadened the conditions supporting liquid-like protoribosomal assemblies. Significance statement: How RNA and peptides first became organized into shared compartments is a central question in the origin of life. Using a model of the ribosome’s ancient catalytic RNA core, we show that the chemical identity of positive charge controls not only whether condensation occurs, but also whether the resulting assemblies are arrested or liquid-like. Heterogeneous peptide populations can form RNA-rich droplets without a single defined RNA-binding sequence; however, Lys- and Arg-containing populations do so more readily than populations containing simpler cationic amino acids. Arginine is especially effective, forming persistent contacts with RNA and preferentially entering the condensed phase. Thus, expansion of cationic amino acid chemistry could have broadened the conditions supporting dynamic peptide– RNA compartments during early ribosome evolution.

Ribosomal proteins contain flexible terminal regions that are averaged out during electron density reconstructions, rendering them absent from experimental models derived by X-ray crystallography or cryogenic electron microscopy. These flexible protein fragments (FPFs) collectively form an invisible coat on the ribosome surface whose presence has been systematically overlooked. Here we analysed FPFs from 36 ribosomes spanning bacteria, eukaryotes, and mitochondria. We found that mitoribosomes harbour the most numerous and longest FPFs. Structural predictions confirmed that FPFs are predominantly disordered across all ribosome classes. Comparison of FPF amino acid composition against proteome-wide background frequencies revealed strong and domain-specific compositional biases. The balance between arginine and lysine content tracks the cardiolipin content of the membrane each ribosome class contacts. The arginine enrichment in mitoribosomal FPFs may additionally reflect selection arising from the RNA-rich environment of mitochondrial RNA granules, membraneless condensates where mitoribosomes are assembled. FPFs are uniformly depleted in aromatic residues, arguing against protein-driven liquid–liquid phase separation propensity. Our findings suggest that the flexibly tethered coat is a highly functional intrinsic part of all ribosomes.

Recently, a tRNATrpCCA with a 4-base-pair (bp) anticodon stem (AS) was shown to efficiently recognize a near-cognate UGA codon in unicellular eukaryotes, such as some trypanosomatids and ciliates, thereby representing a novel codon reassignment mechanism. To determine whether this mechanism also evolved in bacteria, we analysed a dataset of 42 109 genomes, including previously reported cases of stop-to-tryptophan UGA reassignment and a newly identified instance in the phylum Patescibacteriota. We show that the 4-bp AS tRNATrp species are present across diverse bacteria and in some cases likely function in decoding in-frame UGA codons. Most notable is the endosymbiotic bacterium Candidatus Zinderia insecticola, which contains only the near-cognate 4-bp AS tRNATrpCCA, while lacking both canonical 5-bp AS tRNATrpCCA and a tRNATrpUCA. The secondary structure of this 4-bp AS tRNATrp resembles that of its eukaryotic counterpart, suggesting convergent evolution. We experimentally confirmed the UGA readthrough capacity of 4-bp AS tRNATrpCCA in Escherichia coli, and applied molecular dynamics simulations to suggest the underlying mechanism. Furthermore, we tested several predictions based on accepting the previously excluded possibility of C:A base pairing at the 3rd codon position. These findings provide new insights into the structural diversity of transfer RNAs (tRNAs) and expand our understanding of genetic code evolution.

The ribosomal exit tunnel, through which all nascent polypeptides emerge, is formed primarily by ribosomal RNA. Still, ribosomal proteins contribute roughly one quarter of the tunnel walls. In particular, proteins uL4 and uL22 define the narrowest region of the tunnel. This constriction, enriched in basic residues, mediates the earliest protein-protein contacts experienced by a nascent polypeptide. Here, we characterize its conformational dynamics by analyzing 222 Escherichia coli ribosome structures from the Protein Data Bank and by performing unbiased all-atom molecular dynamics simulations of the complete bacterial ribosome with nascent polypeptides of varying length and composition. In simulations of the empty ribosome, the constriction can transiently narrow below the diameter of a water molecule and thus fully occlude the tunnel. At other times it opens wide enough to accommodate a narrow α-helix. The presence of even a short nascent polypeptide shifts the constriction toward wider conformations by approximately 0.2 nm, revealing an adaptive response of the tunnel to its contents. Across all simulated systems, the N-terminal formylmethionine preferentially associates with the tunnel wall at the uL22 side. Our findings replace qualitative description of the tunnel dynamics with a quantitative one. The constriction site functions as a flexible gate, with implications for nascent polypeptide translocation, action of macrolide antibiotics, and the translational ramp.

The study examines how a peptide moves through a carbon nanotube under an applied mechanical force. In particular, using all-atom molecular dynamics simulations it shows the difference between pulling the peptide and pushing it in the same direction.

Ribosomes are critical biomolecular nanomachines responsible for protein synthesis in all known organisms. The function and dynamics of ribosomes can be studied using molecular dynamics computer simulations. Although this task remains challenging at atomic level, several studies have reported all-atom molecular dynamics simulations of the entire ribosome. However, for certain applications, atomistic simulations are impractical due to the limited simulation timescales achievable. In this study, we investigate the applicability of the coarse-grained MARTINI model for simulations of the bacterial ribosome. After testing several simulation setups, we found that the structure of the ribosome and its components are generally well represented compared to the reference experimental structure. Compared with all-atom simulations of the entire ribosome, coarse-grained simulations result in a less flexible and smaller ribosome. We demonstrate how modifications of some parameters of the model can enhance the dynamics of the ribosome to better align with the atomistic model. Our work provides a detailed protocol for coarse-grained simulations of the ribosome and highlights aspects of the model that need improvements.

All proteins in living organisms are produced in ribosomes that facilitate the translation of genetic information into a sequence of amino acid residues. During translation, the ribosome undergoes initiation, elongation, termination, and recycling. In fact, peptide bonds are formed only during the elongation phase, which comprises periodic association of transfer RNAs and multiple auxiliary proteins with the ribosome and the addition of an amino acid to the nascent polypeptide one at a time. The protein spends a considerable amount of time attached to the ribosome. Here, we conceptually divide this portion of the protein lifetime into three stages. We define each stage on the basis of the position of the N‐terminus of the nascent polypeptide within the ribosome exit tunnel and the context of the catalytic center. We argue that nascent polypeptides experience a variety of forces that determine how they translocate through the tunnel and interact with the tunnel walls. We review current knowledge about nascent polypeptide translocation and identify several white spots in our understanding of the birth of proteins.

The ribosome, owing to its exceptional conservation, harbours a remarkable molecular fossil known as the protoribosome. It surrounds the peptidyl transferase center (PTC), responsible for peptide bond formation. While previous studies have demonstrated the PTC activity in RNA alone, our investigation reveals the intricate roles of the ribosomal protein fragments (rPeptides) within the ribosomal core. This research highlights the significance of rPeptides in stability and coacervation of two distinct protoribosomal evolutionary stages. The 617nt ‘big’ protoribosome model, which associates with rPeptides specifically, exhibits a structurally defined and rigid nature, further stabilized by the peptides. In contrast, the 136nt ‘small’ model, previously linked to peptidyltransferase activity, displays greater structural flexibility. While this construct interacts with rPeptides with lower specificity, they induce coacervation of the ‘small’ protoribosome across a wide concentration range, which is concomitantly dependent on the RNA sequence and structure. Moreover, these conditions protect RNA from degradation. This phenomenon suggests a significant evolutionary advantage in the RNA–protein interaction at the early stages of ribosome evolution. The distinct properties of the two protoribosomal stages suggest that rPeptides initially provided compartmentalization and prevented RNA degradation, preceding the emergence of specific RNA–protein interactions crucial for the ribosomal structural integrity.