Mitoribosome structure with cofactors and modifications reveals mechanism of ligand binding and interactions with L1 stalk.

The mitoribosome translates mitochondrial mRNAs and regulates energy conversion that is a signature of aerobic life forms. We present a 2.2 Å resolution structure of human mitoribosome together with validated mitoribosomal RNA (rRNA) modifications, including aminoacylated CP-tRNAVal. The structure shows how mitoribosomal proteins stabilise binding of mRNA and tRNA helping to align it in the decoding center, whereas the GDP-bound mS29 stabilizes intersubunit communication. Comparison between different states, with respect to tRNA position, allowed us to characterize a non-canonical L1 stalk, and molecular dynamics simulations revealed how it facilitates tRNA transitions in a way that does not require interactions with rRNA. We also report functionally important polyamines that are depleted when cells are subjected to an antibiotic treatment. The structural, biochemical, and computational data illuminate the principal functional components of the translation mechanism in mitochondria and provide a description of the structure and function of the human mitoribosome.

Cryo-EM structures of the human Elongator complex at work.

tRNA modifications affect ribosomal elongation speed and co-translational folding dynamics. The Elongator complex is responsible for introducing 5-carboxymethyl at wobble uridine bases (cm5U34) in eukaryotic tRNAs. However, the structure and function of human Elongator remain poorly understood. In this study, we present a series of cryo-EM structures of human ELP123 in complex with tRNA and cofactors at four different stages of the reaction. The structures at resolutions of up to 2.9 Å together with complementary functional analyses reveal the molecular mechanism of the modification reaction. Our results show that tRNA binding exposes a universally conserved uridine at position 33 (U33), which triggers acetyl-CoA hydrolysis. We identify a series of conserved residues that are crucial for the radical-based acetylation of U34 and profile the molecular effects of patient-derived mutations. Together, we provide the high-resolution view of human Elongator and reveal its detailed mechanism of action.

Adaptive evolution: Eukaryotic enzyme's specificity shift to a bacterial substrate.

Prolyl-tRNA synthetase (ProRS), belonging to the family of aminoacyl-tRNA synthetases responsible for pairing specific amino acids with their respective tRNAs, is categorized into two distinct types: the eukaryote/archaeon-like type (E-type) and the prokaryote-like type (P-type). Notably, these types are specific to their corresponding cognate tRNAs. In an intriguing paradox, Thermus thermophilus ProRS (TtProRS) aligns with the E-type ProRS but selectively charges the P-type tRNAPro, featuring the bacterium-specific acceptor-stem elements G72 and A73. This investigation reveals TtProRS's notable resilience to the inhibitor halofuginone, a synthetic derivative of febrifugine emulating Pro-A76, resembling the characteristics of the P-type ProRS. Furthermore, akin to the P-type ProRS, TtProRS identifies its cognate tRNA through recognition of the acceptor-stem elements G72/A73, along with the anticodon elements G35/G36. However, in contrast to the P-type ProRS, which relies on a strictly conserved R residue within the bacterium-like motif 2 loop for recognizing G72/A73, TtProRS achieves this through a non-conserved sequence, RTR, within the otherwise non-interacting eukaryote-like motif 2 loop. This investigation sheds light on the adaptive capacity of a typically conserved housekeeping enzyme to accommodate a novel substrate.

Engineering tRNAs for the Ribosomal Translation of Non-proteinogenic Monomers.

Ribosome-dependent protein biosynthesis is an essential cellular process mediated by transfer RNAs (tRNAs). Generally, ribosomally synthesized proteins are limited to the 22 proteinogenic amino acids (pAAs: 20 l-α-amino acids present in the standard genetic code, selenocysteine, and pyrrolysine). However, engineering tRNAs for the ribosomal incorporation of non-proteinogenic monomers (npMs) as building blocks has led to the creation of unique polypeptides with broad applications in cellular biology, material science, spectroscopy, and pharmaceuticals. Ribosomal polymerization of these engineered polypeptides presents a variety of challenges for biochemists, as translation efficiency and fidelity is often insufficient when employing npMs. In this Review, we will focus on the methodologies for engineering tRNAs to overcome these issues and explore recent advances both in vitro and in vivo. These efforts include increasing orthogonality, recruiting essential translation factors, and creation of expanded genetic codes. After our review on the biochemical optimizations of tRNAs, we provide examples of their use in genetic code manipulation, with a focus on the in vitro discovery of bioactive macrocyclic peptides containing npMs. Finally, an analysis of the current state of tRNA engineering is presented, along with existing challenges and future perspectives for the field.

Activity reconstitution of Kre33 and Tan1 reveals a molecular ruler mechanism in eukaryotic tRNA acetylation.

RNA acetylation is a universal post-transcriptional modification that occurs in various RNAs. Transfer RNA (tRNA) acetylation is found at position 34 (ac4C34) in bacterial tRNAMet and position 12 (ac4C12) in eukaryotic tRNASer and tRNALeu. The biochemical mechanism, structural basis and functional significance of ac4C34 are well understood; however, despite being discovered in the 1960s and identification of Kre33/NAT10 and Tan1/THUMPD1 as modifying apparatuses, ac4C12 modification activity has never been reconstituted for nearly six decades. Here, we successfully reconstituted the ac4C12 modification activity of yeast Kre33 and Tan1. Biogenesis of ac4C12 is primarily dependent on a minimal set of elements, including a canonical acceptor stem, the presence of the 11CCG13 motif and correct D-arm orientation, indicating a molecular ruler mechanism. A single A13G mutation conferred ac4C12 modification to multiple non-substrate tRNAs. Moreover, we were able to introduce ac4C modifications into small RNAs. ac4C12 modification contributed little to tRNA melting temperature and aminoacylation in vitro and in vivo. Collectively, our results realize in vitro activity reconstitution, delineate tRNA substrate selection mechanism for ac4C12 biogenesis and develop a valuable system for preparing acetylated tRNAs as well as non-tRNA RNA species, which will advance the functional interpretation of the acetylation in RNA structures and functions.

From Enigma to Revelation: Unravelling Biological Functions of Ubiquitous Small Ribozymes.

RNA, widely recognized as an information-carrier molecule, is capable of catalyzing essential biological processes through ribozymes. Despite their ubiquity, specific functions in a biological context and phenotypes based on the ribozymes' activity are often unknown. Here, we present the discovery of a subgroup of minimal HDV-like ribozymes, which reside 3' to viral tRNAs and appear to cleave the 3'-trailers of viral premature tRNA transcripts. This proposed tRNA-processing function is unprecedented for any ribozymes, thus, we designate this subgroup as theta ribozymes. Most theta ribozymes were identified in Caudoviricetes bacteriophages, the main constituent (>90%) of the mammalian gut virome. Intriguingly, our findings further suggest the involvement of theta ribozymes in the transition of certain bacteriophages between distinct genetic codes, thus possibly contributing to the phage lysis trigger. Our discovery expands the limited repertoire of biological functions attributed to HDV-like ribozymes and provides insights into the fascinating world of RNA catalysis.

Structural basis of translation inhibition by a valine tRNA-derived fragment.

Translational regulation by non-coding RNAs is a mechanism commonly used by cells to fine-tune gene expression. A fragment derived from an archaeal valine tRNA (Val-tRF) has been previously identified to bind the small subunit of the ribosome and inhibit translation in Haloferax volcanii Here, we present three cryo-electron microscopy structures of Val-tRF bound to the small subunit of Sulfolobus acidocaldarius ribosomes at resolutions between 4.02 and 4.53 Å. Within these complexes, Val-tRF was observed to bind to conserved RNA-interacting sites, including the ribosomal decoding center. The binding of Val-tRF destabilizes helices h24, h44, and h45 and the anti-Shine-Dalgarno sequence of 16S rRNA. The binding position of this molecule partially overlaps with the translation initiation factor aIF1A and occludes the mRNA P-site codon. Moreover, we found that the binding of Val-tRF is associated with steric hindrance of the H69 base of 23S rRNA in the large ribosome subunit, thereby preventing 70S assembly. Our data exemplify how tRNA-derived fragments bind to ribosomes and provide new insights into the mechanisms underlying translation inhibition by Val-tRFs.

Conformational changes of ribosomes during translation elongation resolved by molecular dynamics simulations.

Molecular dynamics simulations have emerged as a powerful set of tools to unravel the intricate dynamics of ribosomes during protein synthesis. Recent advancements in this field have enabled simulations to delve deep into the conformational rearrangements of ribosomes and associated factors, providing invaluable insights into the intricacies of translation. Emphasis on simulations has recently been on translation elongation, such as tRNA selection, translocation, and ribosomal head-swivel motions. These studies have offered crucial structural interpretations of how genetic information is faithfully translated into proteins. This review outlines recent discoveries concerning ribosome conformational changes occurring during translation elongation, as elucidated through molecular dynamics simulations.

Molecular basis of A. thaliana KEOPS complex in biosynthesizing tRNA t6A.

In archaea and eukaryotes, the evolutionarily conserved KEOPS is composed of four core subunits-Kae1, Bud32, Cgi121 and Pcc1, and a fifth Gon7/Pcc2 that is found in fungi and metazoa. KEOPS cooperates with Sua5/YRDC to catalyze the biosynthesis of tRNA N6-threonylcarbamoyladenosine (t6A), an essential modification needed for fitness of cellular organisms. Biochemical and structural characterizations of KEOPSs from archaea, yeast and humans have determined a t6A-catalytic role for Kae1 and auxiliary roles for other subunits. However, the precise molecular workings of KEOPSs still remain poorly understood. Here, we investigated the biochemical functions of A. thaliana KEOPS and determined a cryo-EM structure of A. thaliana KEOPS dimer. We show that A. thaliana KEOPS is composed of KAE1, BUD32, CGI121 and PCC1, which adopts a conserved overall arrangement. PCC1 dimerization leads to a KEOPS dimer that is needed for an active t6A-catalytic KEOPS-tRNA assembly. BUD32 participates in direct binding of tRNA to KEOPS and modulates the t6A-catalytic activity of KEOPS via its C-terminal tail and ATP to ADP hydrolysis. CGI121 promotes the binding of tRNA to KEOPS and potentiates the t6A-catalytic activity of KEOPS. These data and findings provide insights into mechanistic understanding of KEOPS machineries.

Structural insights into the decoding capability of isoleucine tRNAs with lysidine and agmatidine.

The anticodon modifications of transfer RNAs (tRNAs) finetune the codon recognition on the ribosome for accurate translation. Bacteria and archaea utilize the modified cytidines, lysidine (L) and agmatidine (agm2C), respectively, in the anticodon of tRNAIle to decipher AUA codon. L and agm2C contain long side chains with polar termini, but their functions remain elusive. Here we report the cryogenic electron microscopy structures of tRNAsIle recognizing the AUA codon on the ribosome. Both modifications interact with the third adenine of the codon via a unique C-A geometry. The side chains extend toward 3' direction of the mRNA, and the polar termini form hydrogen bonds with 2'-OH of the residue 3'-adjacent to the AUA codon. Biochemical analyses demonstrated that AUA decoding is facilitated by the additional interaction between the polar termini of the modified cytidines and 2'-OH of the fourth mRNA residue. We also visualized cyclic N6-threonylcarbamoyladenosine (ct6A), another tRNA modification, and revealed a molecular basis how ct6A contributes to efficient decoding.

Misacylation of tRNA with Ser-Pro Dipeptide for In Vitro Transcription-Translation.

Serine-proline (Ser-Pro) backbone-modified dipeptide analogues are powerful tools to investigate the role of cis-trans isomerization in the regulation of the cell cycle and transcription. These studies have previously been limited to synthetic peptides, whose synthesis is a challenge for larger peptides due to the compounding yield loss incurred in each step. We now introduce a method for the aminoacylation of tRNA with dipeptides and dipeptide analogs to permit the installation of cis- and trans-locked Ser-Pro analogues into full-length proteins. To that end, we synthesized the 3,5-dinitrobenzyl (DNB)-activated esters of a native Ser-Pro dipeptide and its cis- and trans-locked alkene analogs. Murakami et al. created the DNB flexizyme (dFx), a ribozyme that acylates tRNA with DNB esters of amino acids to permit unnatural amino acids to be incorporated into proteins. A tRNA from yeast that recognizes the amber stop codon, along with the dFx flexizyme, were generated by in vitro transcription with T7 RNA polymerase. dFx was used to successfully catalyze the chemical misacylation of truncated amber tRNA with the Ser-Pro-DNB activated dipeptide. This method allows the introduction of non-native Ser-Pro dipeptide mimics into full-length proteins by in vitro transcription-translation. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Synthesis of 3,5-dinitrobenzyl activated esters of Ser-Pro Basic Protocol 2: Preparation of truncated amber tRNA Basic Protocol 3: Acylation of amber-tRNA by the dFx flexizyme Basic Protocol 4: PAGE electrophoresis of tRNASerPro.

The complete mitochondrial genome of Chibiraga houshuaii (Lepidoptera, Limacodidae) and its phylogenetic implications.

Chibiraga is a mall East Asian genus in the family Limacodidae (slug-moths). The latter includes many agricultural pests. Mitochondrial genome analysis is an important tool for studying insect molecular identification and phylogenetics. However, there are very few mitogenome sequences available for Limacodidae species, and none for the genus Chibiraga at all. To explore the mitogenome features of Chibiraga and verify its phylogenetic position, the complete mitogenome of Chibiraga houshuaii was sequenced and annotated. The complete 15,487 bp genome encoded 37 mitochondrial genes, including 13 protein-coding genes (PCGs), 22 transfer RNA (tRNA) genes, two ribosomal RNA (rRNA) genes, and a control region (CR). Most of the PCGs had typical ATN start codons and terminated with TAA or a single T residue. UUA (Leu2), AUU (Ile), UUU (Phe), AUA (Met) and AAU (Asn) were the five most frequently used codons. All tRNAs were folded into cloverleaf secondary structure, except for trnS1, which lacked the DHU arm. Phylogenetic analyses within the superfamily Zygaenoidea were performed based on multiple datasets from mitochondrial genes. The results showed that the families Phaudidae, Limacodidae and Zygaenidae were respectively recovered as monophyly; C. houshuaii was clustered in a clade with nettle type larvae in Limacodidae.

Chemical manipulation of m1A mediates its detection in human tRNA.

N 1-methyl adenosine (m1A) is a widespread RNA modification present in tRNA, rRNA, and mRNA. m1A modification sites in tRNAs are evolutionarily conserved and its formation on tRNA is catalyzed by methyltransferase TRMT61A and TRMT6 complex. m1A promotes translation initiation and elongation. Due to its positive charge under physiological conditions, m1A can notably modulate RNA structure. It also blocks Watson-Crick-Franklin base-pairing and causes mutation and truncation during reverse transcription. Several misincorporation-based high-throughput sequencing methods have been developed to sequence m1A. In this study, we introduce a reduction-based m1A sequencing (red-m1A-seq). We report that NaBH4 reduction of m1A can improve the mutation and readthrough rates using commercially available RT enzymes to give a better positive signature, while alkaline-catalyzed Dimroth rearrangement can efficiently convert m1A to m6A to provide good controls, allowing the detection of m1A with higher sensitivity and accuracy. We applied red-m1A-seq to sequence human small RNA, and we not only detected all the previously reported tRNA m1A sites, but also new m1A sites in mt-tRNAAsn-GTT and 5.8S rRNA.

Fungi of the order Mucorales express a "sealing-only" tRNA ligase.

Some eukaryotic pre-tRNAs contain an intron that is removed by a dedicated set of enzymes. Intron-containing pre-tRNAs are cleaved by tRNA splicing endonuclease, followed by ligation of the two exons and release of the intron. Fungi use a "heal and seal" pathway that requires three distinct catalytic domains of the tRNA ligase enzyme, Trl1. In contrast, humans use a "direct ligation" pathway carried out by RTCB, an enzyme completely unrelated to Trl1. Because of these mechanistic differences, Trl1 has been proposed as a promising drug target for fungal infections. To validate Trl1 as a broad-spectrum drug target, we show that fungi from three different phyla contain Trl1 orthologs with all three domains. This includes the major invasive human fungal pathogens, and these proteins can each functionally replace yeast Trl1. In contrast, species from the order Mucorales, including the pathogens Rhizopus arrhizus and Mucor circinelloides, have an atypical Trl1 that contains the sealing domain but lacks both healing domains. Although these species contain fewer tRNA introns than other pathogenic fungi, they still require splicing to decode three of the 61 sense codons. These sealing-only Trl1 orthologs can functionally complement defects in the corresponding domain of yeast Trl1 and use a conserved catalytic lysine residue. We conclude that Mucorales use a sealing-only enzyme together with unidentified nonorthologous healing enzymes for their heal and seal pathway. This implies that drugs that target the sealing activity are more likely to be broader-spectrum antifungals than drugs that target the healing domains.

Rational design of oligonucleotides for enhanced in vitro transcription of small RNA.

All kinds of RNA molecules can be produced by in vitro transcription using T7 RNA polymerase using DNA templates obtained by solid-phase chemical synthesis, primer extension, PCR, or DNA cloning. The oligonucleotide design, however, is a challenge to nonexperts as this relies on a set of rules that have been established empirically over time. Here, we describe a Python program to facilitate the rational design of oligonucleotides, calculated with kinetic parameters for enhanced in vitro transcription (ROCKET). The Python tool uses thermodynamic parameters, performs folding-energy calculations, and selects oligonucleotides suitable for the polymerase extension reaction. These oligonucleotides improve yields of template DNA. With the oligonucleotides selected by the program, the tRNA transcripts can be prepared by a one-pot reaction of the DNA polymerase extension reaction and the transcription reaction. Also, the ROCKET-selected oligonucleotides provide greater transcription yields than that from oligonucleotides selected by Primerize, a leading software for designing oligonucleotides for in vitro transcription, due to the enhancement of template DNA synthesis. Apart from over 50 tRNA genes tested, an in vitro transcribed self-cleaving ribozyme was found to have catalytic activity. In addition, the program can be applied to the synthesis of mRNA, demonstrating the wide applicability of the ROCKET software.

Biosynthesis and function of 7-deazaguanine derivatives in bacteria and phages.

SUMMARYDeazaguanine modifications play multifaceted roles in the molecular biology of DNA and tRNA, shaping diverse yet essential biological processes, including the nuanced fine-tuning of translation efficiency and the intricate modulation of codon-anticodon interactions. Beyond their roles in translation, deazaguanine modifications contribute to cellular stress resistance, self-nonself discrimination mechanisms, and host evasion defenses, directly modulating the adaptability of living organisms. Deazaguanine moieties extend beyond nucleic acid modifications, manifesting in the structural diversity of biologically active natural products. Their roles in fundamental cellular processes and their presence in biologically active natural products underscore their versatility and pivotal contributions to the intricate web of molecular interactions within living organisms. Here, we discuss the current understanding of the biosynthesis and multifaceted functions of deazaguanines, shedding light on their diverse and dynamic roles in the molecular landscape of life.

The potential role of differentially expressed tRNA-derived fragments in high glucose-induced podocytes.

The prevalence of diabetic kidney disease (DKD) is increasing annually. Damage to and loss of podocytes occur early in DKD. tRNA-derived fragments (tRFs), originating from tRNA precursors or mature tRNAs, are associated with various illnesses. In this study, tRFs were identified, and their roles in podocyte injury induced by high-glucose (HG) treatment were explored. High-throughput sequencing of podocytes treated with HG was performed to identify differentially expressed tRFs. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed. The expression levels of nephrin, podocin, and desmin were measured in podocytes after overexpression of tRF-1:24-Glu-CTC-1-M2 (tRF-1:24) and concomitant HG treatment. A total of 647 tRFs were identified, and 89 differentially expressed tRFs (|log2FC| ≥ 0.585; p ≤ .05) were identified in the HG group, of which 53 tRFs were downregulated and 36 tRFs were upregulated. The 10 tRFs with the highest differential expression were detected by real-time quantitative polymerase chain reaction (RT-qPCR), and these results were consistent with the sequencing results. GO analysis revealed that the biological process, cellular component, and molecular function terms in which the tRFs were the most enriched were cellular processes, cellular anatomical entities, and binding. KEGG pathway analysis revealed that tRFs may be involved in signaling pathways related to growth hormones, phospholipase D, the regulation of stem cell pluripotency, and T-/B-cell receptors. Overexpression of tRF-1:24, one of the most differentially expressed tRFs, attenuated podocyte injury induced by HG. Thus, tRFs might be potential biomarkers for podocyte injury in DKD.

Cryo-EM structure of the inactive ribosome complex accumulated in chick embryo cells in cold-stress conditions.

Here, we present the high-resolution structure of the Gallus gallus 80S ribosome obtained from cold-treated chicken embryos. The translationally inactive ribosome complex contains elongation factor eEF2 with GDP, SERPINE1 mRNA binding protein 1 (SERBP1) and deacylated tRNA in the P/E position, showing common features with complexes already described in mammals. Modeling of most expansion segments of G. gallus 28S ribosomal RNA allowed us to identify specific features in their structural organization and to describe areas where a marked difference between mammalian and avian ribosomes could shed light on the evolution of the expansion segments. This study provides the first structure of an avian ribosome, establishing a model for future structural and functional studies on the translational machinery in Aves.

Structural determinants for tRNA selective cleavage by RNase 2/EDN.

tRNA-derived fragments (tRFs) have emerged as key players of immunoregulation. Some RNase A superfamily members participate in the shaping of the tRFs population. By comparing wild-type and knockout macrophage cell lines, our previous work revealed that RNase 2 can selectively cleave tRNAs. Here, we confirm the in vitro protein cleavage pattern by screening of synthetic tRNAs, single-mutant variants, and anticodon-loop DNA/RNA hairpins. By sequencing of tRF products, we identified the cleavage selectivity of recombinant RNase 2 with base specificity at B1 (U/C) and B2 (A) sites, consistent with a previous cellular study. Lastly, protein-hairpin complexes were predicted by MD simulations. Results reveal the contribution of the α1, loop 3 and loop 4, and ß6 RNase 2 regions, where residues Arg36/Asn39/Gln40/Asn65/Arg68/Arg132 provide interactions, spanning from P-1 to P2 sites that are essential for anticodon loop recognition. Knowledge of RNase 2-specific tRFs generation might guide new therapeutic approaches for infectious and immune-related diseases.

Adding α,α-disubstituted and ß-linked monomers to the genetic code of an organism.

The genetic code of living cells has been reprogrammed to enable the site-specific incorporation of hundreds of non-canonical amino acids into proteins, and the encoded synthesis of non-canonical polymers and macrocyclic peptides and depsipeptides1-3. Current methods for engineering orthogonal aminoacyl-tRNA synthetases to acylate new monomers, as required for the expansion and reprogramming of the genetic code, rely on translational readouts and therefore require the monomers to be ribosomal substrates4-6. Orthogonal synthetases cannot be evolved to acylate orthogonal tRNAs with non-canonical monomers (ncMs) that are poor ribosomal substrates, and ribosomes cannot be evolved to polymerize ncMs that cannot be acylated onto orthogonal tRNAs-this co-dependence creates an evolutionary deadlock that has essentially restricted the scope of translation in living cells to α-L-amino acids and closely related hydroxy acids. Here we break this deadlock by developing tRNA display, which enables direct, rapid and scalable selection for orthogonal synthetases that selectively acylate their cognate orthogonal tRNAs with ncMs in Escherichia coli, independent of whether the ncMs are ribosomal substrates. Using tRNA display, we directly select orthogonal synthetases that specifically acylate their cognate orthogonal tRNA with eight non-canonical amino acids and eight ncMs, including several ß-amino acids, α,α-disubstituted-amino acids and ß-hydroxy acids. We build on these advances to demonstrate the genetically encoded, site-specific cellular incorporation of ß-amino acids and α,α-disubstituted amino acids into a protein, and thereby expand the chemical scope of the genetic code to new classes of monomers.