Anticodon Stem Research Articles

Structure–function analysis of tRNA t6A-catalysis, assembly and thermostability of Aquifex aeolicus TsaD2B2 tetramer in complex with TsaE

The universal N6-threonylcarbamoyladenosine (t6A) at position 37 of tRNAs is one of core post-transcriptional modifications that are needed for promoting translational fidelity. In bacteria, TsaC utilizes L-threonine, bicarbonate and ATP to generate an intermediate threonylcarbamoyladenylate (TC-AMP), of which the TC-moiety is transferred to N6 atom of tRNA A37 to generate t6A by TsaD with support of TsaB and TsaE. TsaD and TsaB form a TsaDB dimer to which tRNA and TsaE are competitively bound. The catalytic mechanism of TsaD and auxiliary roles of TsaB and TsaE remain to be fully elucidated. In this study, we reconstituted tRNA t6A biosynthesis using recombinant TsaC, TsaD–TsaB and TsaE from thermophilic Aquifex aeolicus and determined crystal structures of apo-form and ADP-bound form of TsaD2B2 tetramer. Our TsaD2B2–TsaE–tRNA model coupled functional validations reveal that the binding of tRNA or TsaE to TsaDB is regulated by C-terminal tail of TsaB and a helical hairpin α1–α2 of TsaD. A. aeolicus TsaD2B2 or TsaDB possesses a basal divalent ion-dependent t6A-catalytic activity that is stimulated by TsaE at the cost of ATP consumption. Our data suggest that binding of TsaE to TsaDB induces conformational changes of α1, α2, α6, α7 and α8 of TsaD and C-terminal tail of TsaB, leading to release of tRNA t6A and AMP. ATP hydrolysis-driven dissociation of TsaE from TsaDB resets an active conformation of TsaDB. Dimerization of thermophilic TsaDB enhances thermostability and promotes t6A-catalytic activity of TsaD2B2–tRNA, of which GC base pairs in anticodon stem are needed for correct folding of thermophilic tRNA at higher temperatures.

N2-methylguanosine and N2, N2-dimethylguanosine in cytosolic and mitochondrial tRNAs

Decoration of cellular RNAs with modified RNA nucleosides is an important layer of gene expression regulation. Throughout the transcriptome, RNA modifications influence the folding, stability and function of RNAs as well as their interactions with RNA-binding proteins. Although first detected more than 50 years ago, the modified nucleosides N2-methylguanosine (m2G) and N2,N2-dimethylguanosine (m22G) have recently come to the fore through the identification and characterization of the human methyltransferases (MTases) responsible for their installation. In tRNAs, m2G and m22G are present at the junctions between the acceptor stem and the D-arm, and the D-arm and the anticodon stem loop. Here, we review the current knowledge on the effects of mono- and di-methylation of N2 of guanosine on base-pairing and provide an overview of m2(2)G sites in cytosolic and mitochondrial tRNAs. We highlight key features of m2G and m22G MTases, and describe how these enzymes specifically recognize their RNA substrates and target nucleosides. We also discuss the impact of these modifications on tRNA functions, their dynamic regulation and their implications in disease.

Structural analysis of noncanonical translation initiation complexes

Translation initiation is a highly regulated, multi-step process that is critical for efficient and accurate protein synthesis. In bacteria, initiation begins when mRNA, initiation factors, and a dedicated initiator fMet-tRNAfMet bind the small (30S) ribosomal subunit. Specific binding of fMet-tRNAfMet in the peptidyl (P) site is mediated by the inspection of the fMet moiety by initiation factor IF2 and of three conserved G-C base pairs in the tRNA anticodon stem by the 30S head domain. Tandem A-minor interactions form between 16S ribosomal RNA nucleotides A1339 and G1338 and tRNA base pairs G30-C40 and G29-C41, respectively. Swapping the G30-C40 pair of tRNAfMet with C-G (called tRNAfMet M1) reduces discrimination against the noncanonical start codon CUG invitro, suggesting crosstalk between the gripping of the anticodon stem and recognition of the start codon. Here, we solved electron cryomicroscopy structures of Escherichia coli 70S initiation complexes containing the fMet-tRNAfMet M1 variant paired to the noncanonical CUG start codon, in the presence or absence of IF2 and the non-hydrolyzable GTP analog GDPCP, alongside structures of 70S initiation complexes containing this tRNAfMet variant paired to the canonical bacterial start codons AUG, GUG, and UUG. We find that the M1 mutation weakens A-minor interactions between tRNAfMet and 16S nucleotides A1339 and G1338, with IF2 strengthening the interaction of G1338 with the tRNA minor groove. These structures suggest how even slight changes to the recognition of the fMet-tRNAfMet anticodon stem by the ribosome can impact the start codon selection.

Defective post-transcriptional modification of tRNA disrupts mitochondrial homeostasis in Leber’s hereditary optic neuropathy

Leber's Hereditary Optic Neuropathy (LHON) is a rare, maternally inherited eye disease, predominantly due to the degeneration of retinal ganglion cells (RGCs). It is associated with a mitochondrial DNA (mtDNA) point mutation. Our previous study identified that the m.15927G>A homoplasmic mutation damaged the highly conserved basepairing (28C-42G) in anticodon stem of tRNAThr, caused deficient t6A modification and significantly decreased efficiency in aminoacylation and steady-state levels of tRNAThr, and led to mitochondrial dysfunction. Meanwhile, mechanisms underlying mtDNA mutations regulate intracellular signaling related to the mitochondrial and cellular integrity are less explored. Here, we manifested that defective nucleotide modification induced by the m.15927G>A mutation interfered with the expression of nuclear genes involved in cytoplasmic proteins essential for oxidative phosphorylation system (OXPHOS), thereby impacting the assemble and integrity of OXPHOS complexes. As a result of these mitochondrial dysfunctions, there was an imbalance in mitochondrial dynamics, particularly distinguished by an increased occurrence of mitochondrial fission. Excessive fission compromised the autophagy process, including initiation phase, formation and maturation of autophagosome. Both Parkin-mediated mitophagy and receptor-dependent mitophagy were significantly impaired in cybrids haboring the m.15927G>A mutation. These changes facilitated intrinsic apoptosis, as indicated by increased cytochrome c release and elevated levels of apoptosis-associated proteins (e.g., BAK, BAX, cleaved caspase 9, cleaved caspase 3, and cleaved PARP) in the mutant cybrids. This study demonstrates that the m.15927G>A mutation contributes to LHON by dysregulating OXPHOS biogenesis, aberrant quality control, increased autophagy, inhibited mitophagy, and abnormal apoptosis.

Initial Amino Acid:Codon Assignments and Strength of Codon:Anticodon Binding.

The ribosome brings 3'-aminoacyl-tRNA and 3'-peptidyl-tRNAs together to enable peptidyl transfer by binding them in two major ways. First, their anticodon loops are bound to mRNA, itself anchored at the ribosomal subunit interface, by contiguous anticodon:codon pairing augmented by interactions with the decoding center of the small ribosomal subunit. Second, their acceptor stems are bound by the peptidyl transferase center, which aligns the 3'-aminoacyl- and 3'-peptidyl-termini for optimal interaction of the nucleophilic amino group and electrophilic ester carbonyl group. Reasoning that intrinsic codon:anticodon binding might have been a major contributor to bringing tRNA 3'-termini into proximity at an early stage of ribosomal peptide synthesis, we wondered if primordial amino acids might have been assigned to those codons that bind the corresponding anticodon loops most tightly. By measuring the binding of anticodon stem loops to short oligonucleotides, we determined that family-box codon:anticodon pairings are typically tighter than split-box codon:anticodon pairings. Furthermore, we find that two family-box anticodon stem loops can tightly bind a pair of contiguous codons simultaneously, whereas two split-box anticodon stem loops cannot. The amino acids assigned to family boxes correspond to those accessible by what has been termed cyanosulfidic chemistry, supporting the contention that these limited amino acids might have been the first used in primordial coded peptide synthesis.

A highly conserved tRNA modification contributes to C. albicans filamentation and virulence.

tRNA modifications play important roles in maintaining translation accuracy in all domains of life. Disruptions in the tRNA modification machinery, especially of the anticodon stem loop, can be lethal for many bacteria and lead to a broad range of phenotypes in baker's yeast. Very little is known about the function of tRNA modifications in host-pathogen interactions, where rapidly changing environments and stresses require fast adaptations. We found that two closely related fungal pathogens of humans, the highly pathogenic Candida albicans and its much less pathogenic sister species, Candida dubliniensis, differ in the function of a tRNA-modifying enzyme. This enzyme, Hma1, exhibits species-specific effects on the ability of the two fungi to grow in the hypha morphology, which is central to their virulence potential. We show that Hma1 has tRNA-threonylcarbamoyladenosine dehydratase activity, and its deletion alters ribosome occupancy, especially at 37°C-the body temperature of the human host. A C. albicans HMA1 deletion mutant also shows defects in adhesion to and invasion into human epithelial cells and shows reduced virulence in a fungal infection model. This links tRNA modifications to host-induced filamentation and virulence of one of the most important fungal pathogens of humans.IMPORTANCEFungal infections are on the rise worldwide, and their global burden on human life and health is frequently underestimated. Among them, the human commensal and opportunistic pathogen, Candida albicans, is one of the major causative agents of severe infections. Its virulence is closely linked to its ability to change morphologies from yeasts to hyphae. Here, this ability is linked-to our knowledge for the first time-to modifications of tRNA and translational efficiency. One tRNA-modifying enzyme, Hma1, plays a specific role in C. albicans and its ability to invade the host. This adds a so-far unknown layer of regulation to the fungal virulence program and offers new potential therapeutic targets to fight fungal infections.

Genetic analysis of translation initiation in bacteria: An initiator tRNA-centric view.

Translation of messenger RNA (mRNA) in bacteria occurs in the steps of initiation, elongation, termination, and ribosome recycling. The initiation step comprises multiple stages and uses a special transfer RNA (tRNA) called initiator tRNA (i-tRNA), which is first aminoacylated and then formylated using methionine and N10 -formyl-tetrahydrofolate (N10 -fTHF), respectively. Both methionine and N10 -fTHF are produced via one-carbon metabolism, linking translation initiation with active cellular metabolism. The fidelity of i-tRNA binding to the ribosomal peptidyl-site (P-site) is attributed to the structural features in its acceptor stem, and the highly conserved three consecutive G-C base pairs (3GC pairs) in the anticodon stem. The acceptor stem region is important in formylation of the amino acid attached to i-tRNA and in its initial binding to the P-site. And, the 3GC pairs are crucial in transiting the i-tRNA through various stages of initiation. We utilized the feature of 3GC pairs to investigate the nuanced layers of scrutiny that ensure fidelity of translation initiation through i-tRNA abundance and its interactions with the components of the translation apparatus. We discuss the importance of i-tRNA in the final stages of ribosome maturation, as also the roles of the Shine-Dalgarno sequence, ribosome heterogeneity, initiation factors, ribosome recycling factor, and coevolution of the translation apparatus in orchestrating a delicate balance between the fidelity of initiation and/or its leakiness to generate proteome plasticity in cells to confer growth fitness advantages in response to the dynamic nutritional states.

Endogenous trans-translation structure visualizes the decoding of the first tmRNA alanine codon.

Ribosomes stall on truncated or otherwise damaged mRNAs. Bacteria rely on ribosome rescue mechanisms to replenish the pool of ribosomes available for translation. Trans-translation, the main ribosome-rescue pathway, uses a circular hybrid transfer-messenger RNA (tmRNA) to restart translation and label the resulting peptide for degradation. Previous studies have visualized how tmRNA and its helper protein SmpB interact with the stalled ribosome to establish a new open reading frame. As tmRNA presents the first alanine codon via a non-canonical mRNA path in the ribosome, the incoming alanyl-tRNA must rearrange the tmRNA molecule to read the codon. Here, we describe cryo-EM analyses of an endogenous Escherichia coli ribosome-tmRNA complex with tRNAAla accommodated in the A site. The flexible adenosine-rich tmRNA linker, which connects the mRNA-like domain with the codon, is stabilized by the minor groove of the canonically positioned anticodon stem of tRNAAla. This ribosome complex can also accommodate a tRNA near the E (exit) site, bringing insights into the translocation and dissociation of the tRNA that decoded the defective mRNA prior to tmRNA binding. Together, these structures uncover a key step of ribosome rescue, in which the ribosome starts translating the tmRNA reading frame.

Lamotrigine compromises the fidelity of initiator tRNA recruitment to the ribosomal P-site by IF2 and the RbfA release from 30S ribosomes in Escherichia coli

ABSTRACT Lamotrigine (Ltg), an anticonvulsant drug, targets initiation factor 2 (IF2), compromises ribosome biogenesis and causes toxicity to Escherichia coli. However, our understanding of Ltg toxicity in E. coli remains unclear. While our in vitro assays reveal no effects of Ltg on the ribosome-dependent GTPase activity of IF2 or its role in initiation as measured by dipeptide formation in a fast kinetics assay, the in vivo experiments show that Ltg causes accumulation of the 17S precursor of 16S rRNA and leads to a decrease in polysome levels in E. coli. IF2 overexpression in E. coli increases Ltg toxicity. However, the overexpression of initiator tRNA (i-tRNA) protects it from the Ltg toxicity. The depletion of i-tRNA or overexpression of its 3GC mutant (lacking the characteristic 3GC base pairs in anticodon stem) enhances Ltg toxicity, and this enhancement in toxicity is synthetic with IF2 overexpression. The Ltg treatment itself causes a detectable increase in IF2 levels in E. coli and allows initiation with an elongator tRNA, suggesting compromise in the fidelity/specificity of IF2 function. Also, Ltg causes increased accumulation of ribosome-binding factor A (RbfA) on 30S ribosomal subunit. Based on our genetic and biochemical investigations, we show that Ltg compromises the function of i-tRNA/IF2 complex in ribosome maturation.

Pressure pushes tRNALys3 into excited conformational states

Conformational dynamics play essential roles in RNA function. However, detailed structural characterization of excited states of RNA remains challenging. Here, we apply high hydrostatic pressure (HP) to populate excited conformational states of tRNALys3, and structurally characterize them using a combination of HP 2D-NMR, HP-SAXS (HP-small-angle X-ray scattering), and computational modeling. HP-NMR revealed that pressure disrupts the interactions of the imino protons of the uridine and guanosine U-A and G-C base pairs of tRNALys3. HP-SAXS profiles showed a change in shape, but no change in overall extension of the transfer RNA (tRNA) at HP. Configurations extracted from computational ensemble modeling of HP-SAXS profiles were consistent with the NMR results, exhibiting significant disruptions to the acceptor stem, the anticodon stem, and the D-stem regions at HP. We propose that initiation of reverse transcription of HIV RNA could make use of one or more of these excited states.

Structural basis for pre-tRNA recognition and processing by the human tRNA splicing endonuclease complex.

Throughout bacteria, archaea and eukarya, certain tRNA transcripts contain introns. Pre-tRNAs with introns require splicing to form the mature anticodon stem loop. In eukaryotes, tRNA splicing is initiated by the heterotetrameric tRNA splicing endonuclease (TSEN) complex. All TSEN subunits are essential, and mutations within the complex are associated with a family of neurodevelopmental disorders known as pontocerebellar hypoplasia (PCH). Here, we report cryo-electron microscopy structures of the human TSEN-pre-tRNA complex. These structures reveal the overall architecture of the complex and the extensive tRNA binding interfaces. The structures share homology with archaeal TSENs but contain additional features important for pre-tRNA recognition. The TSEN54 subunit functions as a pivotal scaffold for the pre-tRNA and the two endonuclease subunits. Finally, the TSEN structures enable visualization of the molecular environments of PCH-causing missense mutations, providing insight into the mechanism of pre-tRNA splicing and PCH.

Structural basis of pre-tRNA intron removal by human tRNA splicing endonuclease

Removal of the intron from precursor-tRNA (pre-tRNA) is essential in all three kingdoms of life. In humans, this process is mediated by the tRNA splicing endonuclease (TSEN) comprising four subunits: TSEN2, TSEN15, TSEN34, and TSEN54. Here, we report the cryo-EM structures of human TSEN bound to full-length pre-tRNA in the pre-catalytic and post-catalytic states at average resolutions of 2.94 and 2.88Å, respectively. Human TSEN features an extended surface groove that holds the L-shaped pre-tRNA. The mature domain of pre-tRNA is recognized by conserved structural elements of TSEN34, TSEN54, and TSEN2. Such recognition orients the anticodon stem of pre-tRNA and places the 3'-splice site and 5'-splice site into the catalytic centers of TSEN34 and TSEN2, respectively. The bulk of the intron sequences makes no direct interaction with TSEN, explaining why pre-tRNAs of varying introns can be accommodated and cleaved. Our structures reveal the molecular ruler mechanism of pre-tRNA cleavage by TSEN.

ThrRS-Mediated Translation Regulation of the RNA Polymerase III Subunit RPC10 Occurs through an Element with Similarity to Cognate tRNA ASL and Affects tRNA Levels.

Aminoacyl tRNA synthetases (aaRSs) are a well-studied family of enzymes with a canonical role in charging tRNAs with a specific amino acid. These proteins appear to also have non-canonical roles, including post-transcriptional regulation of mRNA expression. Many aaRSs were found to bind mRNAs and regulate their translation into proteins. However, the mRNA targets, mechanism of interaction, and regulatory consequences of this binding are not fully resolved. Here, we focused on yeast cytosolic threonine tRNA synthetase (ThrRS) to decipher its impact on mRNA binding. Affinity purification of ThrRS with its associated mRNAs followed by transcriptome analysis revealed a preference for mRNAs encoding RNA polymerase subunits. An mRNA that was significantly bound compared to all others was the mRNA encoding RPC10, a small subunit of RNA polymerase III. Structural modeling suggested that this mRNA includes a stem-loop element that is similar to the anti-codon stem loop (ASL) structure of ThrRS cognate tRNA (tRNAThr). We introduced random mutations within this element and found that almost every change from the normal sequence leads to reduced binding by ThrRS. Furthermore, point mutations at six key positions that abolish the predicted ASL-like structure showed a significant decrease in ThrRS binding with a decrease in RPC10 protein levels. Concomitantly, tRNAThr levels were reduced in the mutated strain. These data suggest a novel regulatory mechanism in which cellular tRNA levels are regulated through a mimicking element within an RNA polymerase III subunit in a manner that involves the tRNA cognate aaRS.

Identification of a novel 5-aminomethyl-2-thiouridine methyltransferase in tRNA modification.

The uridine at the 34th position of tRNA, which is able to base pair with the 3'-end codon on mRNA, is usually modified to influence many aspects of decoding properties during translation. Derivatives of 5-methyluridine (xm5U), which include methylaminomethyl (mnm-) or carboxymethylaminomethyl (cmnm-) groups at C5 of uracil base, are widely conserved at the 34th position of many prokaryotic tRNAs. In Gram-negative bacteria such as Escherichia coli, a bifunctional MnmC is involved in the last two reactions of the biosynthesis of mnm5(s2)U, in which the enzyme first converts cmnm5(s2)U to 5-aminomethyl-(2-thio)uridine (nm5(s2)U) and subsequently installs the methyl group to complete the formation of mnm5(s2)U. Although mnm5s2U has been identified in tRNAs of Gram-positive bacteria and plants as well, their genomes do not contain an mnmC ortholog and the gene(s) responsible for this modification is unknown. We discovered that MnmM, previously known as YtqB, is the methyltransferase that converts nm5s2U to mnm5s2U in Bacillus subtilis through comparative genomics, gene complementation experiments, and in vitro assays. Furthermore, we determined X-ray crystal structures of MnmM complexed with anticodon stem loop of tRNAGln. The structures provide the molecular basis underlying the importance of U33-nm5s2U34-U35 as the key determinant for the specificity of MnmM.

Short tRNA anticodon stem and mutant eRF1 allow stop codon reassignment.

Cognate tRNAs deliver specific amino acids to translating ribosomes according to the standard genetic code, and three codons with no cognate tRNAs serve as stop codons. Some protists havereassigned all stop codons as sense codons, neglecting this fundamental principle1-4. Here we analyse the in-frame stop codons in 7,259 predicted protein-coding genes of a previously undescribed trypanosomatid, Blastocrithidia nonstop. We reveal that in this species in-framestop codons are underrepresented in genes expressed at high levels and that UAA serves as the only termination codon. Whereas new tRNAsGlu fully cognate to UAG and UAA evolved to reassign these stop codons, the UGA reassignment followed a different path through shortening the anticodon stem of tRNATrpCCA from five to four base pairs (bp). The canonical 5-bp tRNATrp recognizes UGG as dictated by the genetic code, whereas its shortened 4-bp variant incorporates tryptophan also into in-frame UGA. Mimicking this evolutionary twist by engineering both variants from B. nonstop, Trypanosoma brucei and Saccharomyces cerevisiae and expressing them in the last two species, we recorded a significantly higher readthrough for all 4-bp variants. Furthermore,a gene encoding B. nonstop release factor 1 acquired a mutation that specifically restricts UGA recognition, robustly potentiating the UGA reassignment. Virtually the same strategy has been adopted by the ciliate Condylostoma magnum. Hence, we describe a previously unknown, universal mechanism that has been exploited in unrelated eukaryotes with reassigned stop codons.

Conservation and Diversification of tRNA t6A-Modifying Enzymes across the Three Domains of Life.

The universal N6-threonylcarbamoyladenosine (t6A) modification occurs at position 37 of tRNAs that decipher codons starting with adenosine. Mechanistically, t6A stabilizes structural configurations of the anticodon stem loop, promotes anticodon-codon pairing and safeguards the translational fidelity. The biosynthesis of tRNA t6A is co-catalyzed by two universally conserved protein families of TsaC/Sua5 (COG0009) and TsaD/Kae1/Qri7 (COG0533). Enzymatically, TsaC/Sua5 protein utilizes the substrates of L-threonine, HCO3-/CO2 and ATP to synthesize an intermediate L-threonylcarbamoyladenylate, of which the threonylcarbamoyl-moiety is subsequently transferred onto the A37 of substrate tRNAs by the TsaD-TsaB -TsaE complex in bacteria or by the KEOPS complex in archaea and eukaryotic cytoplasm, whereas Qri7/OSGEPL1 protein functions on its own in mitochondria. Depletion of tRNA t6A interferes with protein homeostasis and gravely affects the life of unicellular organisms and the fitness of higher eukaryotes. Pathogenic mutations of YRDC, OSGEPL1 and KEOPS are implicated in a number of human mitochondrial and neurological diseases, including autosomal recessive Galloway-Mowat syndrome. The molecular mechanisms underscoring both the biosynthesis and cellular roles of tRNA t6A are presently not well elucidated. This review summarizes current mechanistic understandings of the catalysis, regulation and disease implications of tRNA t6A-biosynthetic machineries of three kingdoms of life, with a special focus on delineating the structure-function relationship from perspectives of conservation and diversity.

Probing the Substrate Requirements of the In Vitro Geranylation Activity of Selenouridine Synthase (SelU).

Natural RNA modifications diversify the structures and functions of existing nucleic acid building blocks. Geranyl is one of the most hydrophobic groups recently identified in bacterial tRNAs. Selenouridine synthase (SelU, also called mnmH) is an enzyme with a dual activity which catalyzes selenation and geranylation in tRNAs containing 2-thiouridine using selenophosphate or geranyl-pyrophosphate as cofactors. In this study, we explored the in vitro geranylation process of tRNA anticodon stem loops (ASL) mediated by SelU and showed that the geranylation activity was abolished when U35 was mutated to A35 (ASL-tRNALys (s2U)UU to ASL-tRNAIle (s2U)AU ). By examining the SelU cofactor geranyl-pyrophosphate (gePP) and its analogues, we found that only the geranyl group, but not dimethylallyl- and farnesyl-pyrophosphate with either shorter or longer terpene chains, could be incorporated into ASL. The degree of tRNA geranylation in the end-point analysis for SelU follows the order of ASLLys (s2UUU) ASLGln (s2UUG) >ASLGlu (s2UUC) . These findings suggest a putative mechanism for substrate discrimination by SelU and reveal key factors that might influence its enzymatic activity. Given that SelU plays an important role in bacterial translation systems, inhibiting this enzyme and targeting its geranylation and selenation pathways could be exploited as a promising strategy to develop SelU-based antibiotics.

Enhanced Codon-Anticodon Interaction at In-Frame UAG Stop Codon Improves the Efficiency of Non-Natural Amino Acid Mutagenesis.

The introduction of non-natural amino acids into proteins through the stop codon readthrough methodology has been used to design proteins for diverse applications. However, this method suffers from low yields of the modified protein, as the suppressor tRNA that recognizes the stop codon is unable to compete effectively with release factor 1 (RF1), which terminates translation. We reasoned that a suppressor tRNA with improved interaction with the UAG stop codon on the mRNA will be able to compete more effectively with RF1. To test this idea, we inserted two 2,6-diaminopurine (D) units in the tRNA anticodon stem loop, including one at the third position of the tRNA anticodon. The modified suppressor tRNA could potentially form additional H-bonds between the N2-exocyclic amine of D and the C2 carbonyl group of uracil, thereby enhancing mRNA-tRNA interaction and/or altering tRNA conformation. The stronger interaction at the codon-anticodon interface resulted in improved UAG decoding efficiency and a higher yield of the modified protein containing a non-natural amino acid at multiple sites. Our findings are consistent with the importance of hydrogen bonding and tRNA conformation at the tRNA-mRNA duplex interface during in-frame UAG suppression, which improves protein translation at multiple UAG stop sites. This work provides valuable inputs toward improved non-natural amino acid mutagenesis for creating designer proteins.

An Alternative Role of RluD in the Fidelity of Translation Initiation in Escherichia coli

The fidelity of initiator tRNA (i-tRNA) selection in the ribosomal P-site is a key step in translation initiation. The highly conserved three consecutive G:C base pairs (3GC pairs) in the i-tRNA anticodon stem play a crucial role in its selective binding in the P-site. Mutations in the 3GC pairs (3GC mutant) render the i-tRNA inactive in initiation. Here, we show that a mutation (E265K) in the unique C-terminal tail domain of RluD, a large ribosomal subunit pseudouridine synthase, results in compromised fidelity of initiation and allows initiation with the 3GC mutant i-tRNA. RluD modifies the uridine residues in H69 to pseudouridines. However, the role of its C-terminal tail domain remained unknown. The E265K mutation does not diminish the pseudouridine synthase activity of RluD, or the growth phenotype of Escherichia coli, or cause any detectable defects in the ribosomal assembly in our assays. However, in our in vivo analyses, we observed that the E265K mutation resulted in increased retention of the ribosome binding factor A (RbfA) on 30S suggesting a new role of RluD in contributing to RbfA release, a function which may be attributed to its (RluD) C-terminal tail domain. The studies also reveal that deficiency of RbfA release from 30S compromises the fidelity of i-tRNA selection in the ribosomal P-site.

TsRNAs and their regulation of gene expression in plants in response to abiotic stress

<p indent="0mm">Transfer RNA (tRNA)-derived small RNAs (tsRNAs) are an emerging class of conserved non-coding RNAs that are usually produced from precursor tRNAs (pre-tRNAs) or mature tRNAs cleavage by endonuclease which occurs under stress conditions.There are mainly two types of tsRNAs: Namely tRNA-derived stress-induced RNAs (tiRNAs), also known as tRNA halves (tRHs), and tRNA-derived fragments (tRFs), which differ in the cleavage sites of mature tRNAs or pre-tRNAs. tiRNAs (including 5′ and 3′-tiRNAs) with <sc>30–40 nt</sc> in length are generated by ribonuclease A (RNase A) or ribonuclease T2 cleavage in the anticodon-loop of mature tRNA through a Dicer-independent pathway. tRFs (including 5-tRFs, 3-tRFs, 1-tRFs, 2-tRFs, and i-tRFs) are derived from pre-tRNAs or mature tRNAs through both Dicer-dependent and Dicer-independent pathways. 5-tRFs are generated through the endonucleolytic cleavage in the D-loop or in the region between D-loop and anticodon-loop of mature tRNAs by Dicer or RNase T2. 3-tRFs are produced from the cleavage in the T-loop by Dicer or RNase A. 1-tRFs with a 3′ poly-U are generated by RNase Z cleavage in the 3′ trailer of pre-tRNA during tRNA maturation. 2-tRFs generated by unknown ribonuclease contain the complete anticodon-loop and anticodon stem. i-tRFs produced by unknown ribonuclease are derived from the internal region of mature tRNA, including the anticodon-loop and part of D/T-loop. Based on the origins and types of tsRNAs, tsRNAs are named according to a naming convention in the form of X-tsRNA^AA-NNN (tsRNA represents the species of tiRNAs and tRFs; X represents tsRNAs subtypes of 1, 2, 3, and 5; AA represents the abbreviation of amino acid carried by the mapped tRNAs; NNN represents the anticodon of the mapped tRNAs). tsRNAs exist widely in prokaryotes and eukaryotes, exhibiting spatiotemporally specific expression pattern to function properly, while their dysregulation triggers homeostatic imbalance. Accumulating evidence has demonstrated that tsRNAs play important roles in various biological processes mainly through the regulation of gene expression at transcriptional, post-transcriptional and translational levels. Therefore, exploring the biological function and molecular mechanism of tsRNAs has become a new research hotspot in the field of non-coding small RNAs. Here, we briefly review the advances in the classification, biogenesis, characteristics, and biological function of tsRNAs, with a focus on the regulating roles of tsRNAs in gene expression of plants in response to abiotic stress. Different types of tsRNAs induced by abiotic stresses, such as oxidative stress, phosphate starvation, drought, UV-radiation, salt stress, heat and cold-stress, were summarized in various plants. New sequencing technologies, including tRNA-seq (also known as ARM-seq), cP-RNA-seq (2′,3′-cyclic phosphate RNA sequencing beneficial for tRNA halves), YAMAT-seq (an efficient and convenient method for high-throughput sequencing of mature tRNAs), PANDORA-seq (uncovering small RNAs with different termini and nucleoside methylation), and RtcB sRNA-seq (an RtcB ligation-based small RNA sequencing method to profile 3′-cP-terminated and 3′-OH-terminated 5′-tiRNAs and 5-tRFs), have been developed to characterize plant tsRNAs more efficiently. Simultaneously, a variety of bioinformatics tools and Web databases have been developed for analyzing plant tsRNAs, such as tRex (an online repository of <italic>Arabidopsis</italic> tsRNAs), PtRFdb (a collection of over 5000 unique tsRNAs from 10 plant species), tRFanalyzer (for spatiotemporally analyzing the expression of tRNAs and tsRNAs in <italic>Arabidopsis</italic> and rice), and tsRBase (a comprehensive database of over 120000 sequences, expression features and function of tsRNAs from 20 species including five plant species, <italic>Arabidopsis thaliana</italic>, <italic>Oryza sativa</italic>, <italic>Zea mays</italic>, <italic>Glycine max </italic>and<italic> Vitis vinifera</italic>). The improvement of next-generation sequencing (NGS) technologies has accelerated the quantification of tsRNA populations in plants, contributing greatly for further functional studies of plant tsRNAs.