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[Display omitted] • Designer translating organelles called OTOs enable mRNA-specific protein engineering. • Different RS/tRNA pairs were tested within OTOs and found to impair OTO functionality. • Chimeric PheRS/tRNAPhe pair was optimized to restore the correct OTO performance. Site-specific incorporation of noncanonical amino acids (ncAAs) can be realized by genetic code expansion (GCE) technology. Different orthogonal tRNA synthetase/tRNA (RS/tRNA) pairs have been developed to introduce a ncAA at the desired site, delivering a wide variety of functionalities that can be installed into selected proteins. Cytoplasmic expression of RS/tRNA pairs can cause a problem with background ncAA incorporation into host proteins. The application of orthogonally translating organelles (OTOs), inspired by the concept of phase separation, provides a solution for this issue in mammalian cells, allowing site-specific and protein-selective ncAA incorporation. So far, only Methanosarcina mazei (Mm) pyrrolysyl-tRNA synthetase (PylRS) has been used within OTOs, limiting the method's potential. Here, we explored the implementation of four other widely used orthogonal RS/tRNA pairs with OTOs, which, to our surprise, were unsuccessful in generating mRNA-selective GCE. Next, we tested several experimental solutions and developed a new chimeric phenylalanyl-RS/tRNA pair that enables ncAA incorporation in OTOs in a site-specific and protein-selective manner. Our work reveals unaccounted design constraints in the spatial engineering of enzyme functions using designer organelles and presents a strategy to overcome those in vivo. We then discuss current limitations and future directions of in-cell engineering in general and protein engineering using GCE specifically. [ABSTRACT FROM AUTHOR]

Topoisomerases maintain topological homeostasis of bacterial chromosomes by catalysing changes in DNA linking number. The resolution of RNA entanglements occurring in the cell would also require catalytic action of topoisomerases. We describe RNA topoisomerase and hydrolysis activities in DNA topoisomerase I (topo I) from mycobacteria. The interaction of topo I with mRNA, tRNA and rRNA suggested its role in some aspect of RNA metabolism; the enzyme participates in rRNA maturation via its RNA hydrolysis activity. Accumulation of rRNA precursors in a topo I knockdown strain and the rescue of rRNA processing deficiency in RNaseE knockdown cells by topo I expression indicated the enzyme's back-up support to RNases involved in rRNA processing. We demonstrate that the active-site tyrosine of the enzyme mediates catalytic reactions with both DNA/RNA substrates, and RNA topoisomerase activity can follow two reaction paths in contrast to its DNA topoisomerase activity. Mutation in the canonical proton relay pathway impacts DNA topoisomerase activity whilst retaining activity on RNA substrates. The mycobacterial topo I thus exemplifies the resourcefulness and parsimony of biological catalysis in harnessing the limited chemical repertoire at its disposal to find common solutions to mechanistically related challenges of phosphodiester breakage/exchange reactions in DNA and RNA that are essential for cell survival. Unlabelled Image • A type IA DNA topoisomerase with RNA topoisomerase and hydrolysis activity • First analysis of all the steps of RNA topoisomerase activity viz. RNA binding, cleavage, strand passage and religation; an assay for RNA strand passage step • First analysis of the reaction mechanism for RNA topoisomerisation. Two reaction paths for RNA topoisomerisation: proton relay route and 2′-OH group catalysed reaction. • Active-site tyrosine required for all the catalytic steps with DNA and RNA; pH-dependent switch from RNA topoisomerisation to hydrolysis • A new function for type IA topoisomerase–rRNA processing activity, a backup role to rRNA processing machinery [ABSTRACT FROM AUTHOR]

One-carbon metabolism produces methionine and N10-formyl-tetrahydrofolate (N10-fTHF) required for aminoacylation and formylation of initiator tRNA (i-tRNA), respectively. In Escherichia coli , N10-fTHF is made from 5, 10-methylene-THF by a two-step reaction using 5,10-methylene-THF dehydrogenase/cyclohydrolase (FolD). The i-tRNAs from all domains of life possess a highly conserved sequence of three consecutive G–C base pairs (3GC pairs) in their anticodon stem. A 3GC mutant i-tRNA (wherein the 3GC pairs are mutated to those found in elongator tRNAMet) is incompetent in initiation in E. coli (even though it is efficiently aminoacylated and formylated). Here, we show that E. coli strains having mutations in FolD (G122D or C58Y or P140L) allow a plasmid encoded 3GC mutant i-tRNA to participate in initiation. In vitro , the FolD mutants are highly compromised in their dehydrogenase/cyclohydrolase activities leading to reduced production of N10-fTHF and decreased rates of i-tRNA formylation. The perturbation of one-carbon metabolism by trimethoprim (inhibitor of dihydrofolate reductase) phenocopies FolD deficiency and allows initiation with the 3GC mutant i-tRNA. This study reveals an important crosstalk between one-carbon metabolism and the fidelity of translation initiation via formylation of i-tRNA, and suggests that augmentation of the age old sulfa drugs with FolD inhibitors could be an important antibacterial strategy. Unlabelled Image • Met and N10-fTHF are used to convert tRNAfMet to fMet-tRNAfMet. • Metabolic changes in N10-fTHF levels regulate fidelity of translation initiation. • Deficiency of N10-fTHF results in participation of tRNAfMet at the step of elongation. • Changes in N10-fTHF levels provide important cellular strategy for growth fitness. • Use of sulfa drugs with the FolD inhibitors could serve as potent antibacterials. [ABSTRACT FROM AUTHOR]

Genetic code expansion (GCE) technologies incorporate non-canonical amino acids (ncAAs) into proteins at amber stop codons. To avoid unwanted truncated protein and improve ncAA–protein yields, genomically recoded strains of Escherichia coli lacking Release Factor 1 (RF1) are becoming increasingly popular expression hosts for GCE applications. In the absence of RF1, however, endogenous near-cognate amber suppressing tRNAs can lead to contaminating protein forms with natural amino acids in place of the ncAA. Here, we show that a second-generation amino-acyl tRNA synthetase (aaRS)/tRNA CUA pair for site-specific incorporation of 3-nitro-tyrosine could not outcompete near-cognate suppression in an RF1-deficient expression host and therefore could not produce homogenously nitrated protein. To resolve this, we used Rosetta to target positions in the nitroTyr aaRS active site for improved substrate binding, and then constructed of a small library of variants to subject to standard selection protocols. The top selected variant had an ~ 2-fold greater efficiency, and remarkably, this relatively small improvement enabled homogeneous incorporation of nitroTyr in an RF1-deficient expression host and thus eliminates truncation issues associated with typical RF1-containing expression hosts. Structural and biochemical data suggest the aaRS efficiency improvement is based on higher affinity substrate binding. Taken together, the modest improvement in aaRS efficiency provides a large practical impact and expands our ability to study the role protein nitration plays in disease development through producing homogenous, truncation-free nitroTyr-containing protein. This work establishes Rosetta-guided design and incremental aaRS improvement as a viable and accessible path to improve GCE systems challenged by truncation and/or near-cognate suppression issues. Unlabelled Image • Near-cognate amber codon suppression in "recoded" E. coli expression hosts that lack Release Factor 1 creates a general fidelity problem for genetic code expansion (GCE). • Improving the efficiency of an amino-acyl tRNA synthetase/tRNA pair overcame these near-cognate suppression problems. • Improvements were made using Rosetta-guided library design and directed evolution strategies. • This new 3-nitro-tyrosine GCE system expands our ability to produce site-specifically nitrated proteins in E. coli. [ABSTRACT FROM AUTHOR]

Protein expression in multicellular organisms varies widely across tissues. Codon usage in the transcriptome of each tissue is derived from genomic codon usage and the relative expression level of each gene. We created a comprehensive computational resource that houses tissue-specific codon, codon-pair, and dinucleotide usage data for 51 Homo sapiens tissues (TissueCoCoPUTs: https://hive.biochemistry.gwu.edu/review/tissue%5fcodon), using transcriptome data from the Broad Institute Genotype-Tissue Expression (GTEx) portal. Distances between tissue-specific codon and codon-pair frequencies were used to generate a dendrogram based on the unique patterns of codon and codon-pair usage in each tissue that are clearly distinct from the genomic distribution. This novel resource may be useful in unraveling the relationship between codon usage and tRNA abundance, which could be critical in determining translation kinetics and efficiency across tissues. Areas of investigation such as biotherapeutic development, tissue-specific genetic engineering, and genetic disease prediction will greatly benefit from this resource. Image 1 • New database based on tissue-specific GTEx transcriptomic data for 51 Homo sapiens tissues. • User-friendly interface visualizing codon, codon-pair, and dinucleotide data. • TissueCoCoPUTs can be used in predicting translation efficiency and kinetics. • TissueCoCoPUTs can be used for tissue-targeted recombinant therapeutics or gene therapy design and optimization. • TissueCoCoPUTs may facilitate the development of genetic disease prediction tools. [ABSTRACT FROM AUTHOR]

Selection of correct aminoacyl (aa)-tRNA at the ribosomal A site is fundamental to maintaining translational fidelity. Aa-tRNA selection is a multistep process facilitated by the guanosine triphosphatase elongation factor (EF)-Tu. EF-Tu delivers aa-tRNA to the ribosomal A site and participates in tRNA selection. The structural mechanism of how EF-Tu is involved in proofreading remains to be fully resolved. Here, we provide evidence that switch I of EF-Tu facilitates EF-Tu's involvement during aa-tRNA selection. Using structure-based and explicit solvent molecular dynamics simulations based on recent cryo-electron microscopy reconstructions, we studied the conformational change of EF-Tu from the guanosine triphosphate to guanine diphosphate conformation during aa-tRNA accommodation. Switch I of EF-Tu rapidly converts from an α-helix into a β-hairpin and moves to interact with the acceptor stem of the aa-tRNA. In doing so, switch I gates the movement of the aa-tRNA during accommodation through steric interactions with the acceptor stem. Pharmacological inhibition of the aa-tRNA accommodation pathway prevents the proper positioning of switch I with the aa-tRNA acceptor stem, suggesting that the observed interactions are specific for cognate aa-tRNA substrates, and thus capable of contributing to the fidelity mechanism. Image 1 • Approximate energy landscapes identify switch I barrier for tRNA accommodation. • Switch I interactions with tRNA are required for accommodation. • tRNA adopts metastable state in accommodation, consistent with single molecule data. • Pharmacological inhibition of accommodation alters interactions between switch I and tRNA. [ABSTRACT FROM AUTHOR]

Brain Expressed X-linked (BEX) protein family consists of five members in humans and is highly expressed during neuronal development. They are known to participate in cell cycle and in signaling pathways involved in neurodegeneration and cancer. BEX3 possess a conserved leucine-rich nuclear export signal and experimental data confirmed BEX3 nucleocytoplasmic shuttling. Previous data revealed that mouse BEX3 auto-associates in an oligomer rich in intrinsic disorder. In this work, we show that human BEX3 (hBEX3) has well-defined three-dimensional structure in the presence of small fragments of tRNA (tRFs). Conversely, the nucleic acids-free purified hBEX3 presented disordered structure. Small-angle X-ray scattering data revealed that in the presence of tRFs, hBEX3 adopts compact globular fold, which is very distinct from the elongated high-order oligomer formed by the pure protein. Furthermore, microscopy showed that hBEX3 undergoes condensation in micron-sized protein-rich droplets in vitro. In the presence of tRFs, biomolecular condensates were smaller and in higher number, showing acridine orange green fluorescence emission, which corroborated with the presence of base-paired nucleic acids. Additionally, we found that over time hBEX3 transits from liquid condensates to aggregates that are reversible upon temperature increment and dissolved by 1,6-hexanediol. hBEX3 assemblies display different morphology in the presence of the tRFs that seems to protect from amyloid formation. Collectively, our findings support a role for tRFs in hBEX3 disorder-to-order transition and modulation of phase transitions. Moreover, hBEX3 aggregation-prone features and the specificity in interaction with tRNA fragments advocate paramount importance toward understanding BEX family involvement in neurodevelopment and cell death. Unlabelled Image [ABSTRACT FROM AUTHOR]

Three of six arginine codons (CGU, CGC, and CGA) are decoded by two Escherichia coli tRNAArg isoacceptors. The anticodon stem and loop (ASL) domains of tRNAArg1 and tRNAArg2 both contain inosine and 2-methyladenosine modifications at positions 34 (I 34) and 37 (m2A 37). tRNAArg1 is also modified from cytidine to 2-thiocytidine at position 32 (s2C 32). The s2C 32 modification is known to negate wobble codon recognition of the rare CGA codon by an unknown mechanism, while still allowing decoding of CGU and CGC. Substitution of s2C 32 for C 32 in the Saccharomyces cerevisiae tRNAIle IAU anticodon stem and loop domain (ASL) negates wobble decoding of its synonymous A-ending codon, suggesting that this function of s2C at position 32 is a generalizable property. X-ray crystal structures of variously modified ASLArg1 ICG and ASLArg2 ICG constructs bound to cognate and wobble codons on the ribosome revealed the disruption of a C 32- A 38 cross-loop interaction but failed to fully explain the means by which s2C 32 restricts I 34 wobbling. Computational studies revealed that the adoption of a spatially broad inosine-adenosine base pair at the wobble position of the codon cannot be maintained simultaneously with the canonical ASL U-turn motif. C 32- A 38 cross-loop interactions are required for stability of the anticodon/codon interaction in the ribosomal A-site. Image 1 • s2C 32 of tRNAArg's ASL negates wobble position I binding to codons beginning in A. • s2C 32 alters C 32- A 38 cross-loop interactions so that I 34 /A1 cannot be maintained. • The negative effect of s2C 32 on A3 decoding may be an advantage in some environs. [ABSTRACT FROM AUTHOR]

Aminoacyl-tRNA synthetases (AARSs) ligate amino acids to their cognate tRNAs during protein synthesis. In humans, eight AARSs and three non-enzymatic AARS-interacting multifunctional proteins (AIMP1–3), which are involved in various biological processes, form a multi-tRNA synthetase complex (MSC). Elucidation of the structures and multiple functions of individual AARSs and AIMPs has aided current understanding of the structural arrangement of MSC components and their assembly processes. Here, we report the crystal structure of a complex comprising a motif from aspartyl-tRNA synthetase (DRS) and the glutathione transferase (GST)-homology domains of methionyl-tRNA synthetase (MRS), glutamyl-prolyl-tRNA synthetase (EPRS), AIMP2, and AIMP3. In the crystal structure, the four GST domains are assembled in the order of MRS-AIMP3-EPRS-AIMP2, and the GST domain of AIMP2 binds DRS through the β-sheet in the GST domain. The C-terminus of AIMP3 enhances the binding of DRS to the tetrameric GST complex. A DRS dimer and two GST tetramers binding to the dimer with 2-fold symmetry complete a decameric complex. The formation of this complex enhances the stability of DRS and enables it to retain its reaction intermediate, aspartyl adenylate. Since the catalytic domains of MRS and EPRS are connected to the decameric complex through their flexible linker peptides, and lysyl-tRNA synthetase and AIMP1 are also linked to the complex via the N-terminal region of AIMP2, the DRS–GST tetramer complex functions as a frame in the MSC. Unlabelled Image [ABSTRACT FROM AUTHOR]

Transcription-coupled repair is mediated by the Mfd protein. TCR is defined as the preferential repair of DNA lesions in the transcribed strand of actively transcribed genes, and is opposed to the strand-aspecific global genome repair. The Mfd protein mediates TCR by binding to and displacing RNA polymerase, which is stalled at a DNA lesion on the transcribed strand of DNA, then recruiting UvrA and UvrB. The repair cascade results in the recruitment of, and DNA excision by, UvrC; removal of the damage-bearing oligonucleotide by UvrD; "filling-in" of the DNA by DNA polymerase; and sealing of the strands by DNA ligase. The gene required for Mfd was originally identified as a gene needed for the "mutation frequency decline" phenotype in which the repair of certain UV-induced lesions in the transcribed strand of tRNA genes is increased when cells are forced to delay replication immediately following UV exposure. This review will focus on the genetics that led to the discovery of the Mfd gene; summarize the subsequent biochemical, structural and single-molecule interrogations of the Mfd protein; and explore the more recent findings of Mfd in mutagenesis. Unlabelled Image • Mfd enacts transcription-coupled repair in bacteria by remodeling stalled RNAP. • Genetic observations also indicate that Mfd can also increase the rate of evolution of bacteria subjected to antibiotic stress. • We propose that Mfd association with actively transcribing RNAP may result in formation of a negatively supercoiled domain behind the RNAP, which could be a substrate for R-loop formation. • Genetic instability resulting from R-loop formation may in turn help bacteria increase their rate of mutation as they seek a way to become antibiotic resistant. [ABSTRACT FROM AUTHOR]

Abstract Dysregulation of protein translation is emerging as a unifying mechanism in the pathogenesis of many neuronal disorders. Ribosomal RNA (rRNA) and transfer RNA (tRNA) are structural molecules that have complementary and coordinated functions in protein synthesis. Defects in both rRNAs and tRNAs have been described in mammalian brain development, neurological syndromes, and neurodegeneration. In this review, we present the molecular mechanisms that link aberrant rRNA and tRNA transcription, processing and modifications to translation deficits, and neuropathogenesis. We also discuss the interdependence of rRNA and tRNA biosynthesis and how their metabolism brings together proteotoxic stress and impaired neuronal homeostasis. Graphical Abstract Unlabelled Image Highlights • rRNA and tRNA synthesis and functions are controlled by cellular stress conditions. • rRNA and tRNA biosynthesis are interdependent. • Aberrant rRNA and tRNA biogenesis are associated with neurological disorders. [ABSTRACT FROM AUTHOR]

Abstract The accurate modification of the tRNAIle anticodon wobble cytosine 34 is critical for AUA decoding in protein synthesis. Archaeal tRNAIle2 cytosine 34 is modified with agmatine in the presence of ATP by TiaS (tRNAIle2 agmatidine synthetase). However, no structure of apo-form full-length TiaS is available currently. Here, the crystal structures of apo TiaS and a complex of TiaS–agmatine–AMPPCP–Mg are presented, with properly folded zinc ribbon and Cys4-zinc coordination identified. Compared with tRNAIle2-bound form, the architecture of apo TiaS shows a totally different conformation of zinc ribbon. Molecular dynamics simulations of the docking complex between free-state TiaS and tRNAIle2 suggest that zinc ribbon domain is capable of performing large-scale motions to sample substrate binding-competent conformation. Principle component analysis and normal mode analysis show consistent results about the relative directionality of functionally correlated zinc ribbon motions. Apo TiaS and TiaS–agmatine–AMPPCP–Mg/TiaS–AMPCPP–Mg complex structures capture two snapshots of the flexible ATP-Mg binding p2loop step-by-step stabilization. Research from this study provides new insight into TiaS functional mechanism and the dynamic feature of zinc ribbons. Graphical Abstract Unlabelled Image Highlights • TiaS structure identified properly folded fourth domain as Cys4 zinc ribbon. • TiaS showed a totally different conformation from tRNAile2-bound state. • Computational structural analysis—molecular dynamic simulation and normal mode analysis—suggested functionally correlated direction preferences of zinc ribbon motions. • Alternative mechanism of catalysis by TiaS and implications for applied research were discussed. [ABSTRACT FROM AUTHOR]

The Escherichia coli homodimeric proteins MnmE and MnmG form a functional complex, MnmEG, that modifies tRNAs using GTP, methylene-tetrahydrofolate, FAD, and glycine or ammonium. MnmE is a tetrahydrofolate- and GTP-binding protein, whereas MnmG is a FAD-binding protein with each protomer composed of the FAD-binding domain, two insertion domains, and the helical C-terminal domain. The detailed mechanism of the MnmEG-mediated reaction remains unclear partially due to incomplete structural information on the free- and substrate-bound forms of the complex. In this study, we show that MnmG can adopt in solution a dimer arrangement (form I) different from that currently considered as the only biologically active (form II). Normal mode analysis indicates that form I can oscillate in a range of open and closed conformations. Using isothermal titration calorimetry and native red electrophoresis, we show that a form-I open conformation, which can be stabilized in vitro by the formation of an interprotomer disulfide bond between the catalytic C277 residues, appears to be involved in the assembly of the MnmEG catalytic center. We also show that residues R196, D253, R436, R554 and E585 are important for the stabilization of form I and the tRNA modification function. We propose that the form I dynamics regulates the alternative access of MnmE and tRNA to the MnmG FAD active site. Finally, we show that the C-terminal region of MnmG contains a sterile alpha motif domain responsible for tRNA–protein and protein–protein interactions. [ABSTRACT FROM AUTHOR]

The N-terminally myristoylated matrix (MA) domain of the HIV-1 Gag polyprotein promotes virus assembly by targeting Gag to the inner leaflet of the plasma membrane. Recent studies indicate that, prior to membrane binding, MA associates with cytoplasmic tRNAs (including tRNA Lys3 ), and in vitro studies of tRNA-dependent MA interactions with model membranes have led to proposals that competitive tRNA interactions contribute to membrane discrimination. We have characterized interactions between native, mutant, and unmyristylated (myr-) MA proteins and recombinant tRNA Lys3 by NMR spectroscopy and isothermal titration calorimetry. NMR experiments confirm that tRNA Lys3 interacts with a patch of basic residues that are also important for binding to the plasma membrane marker, phosphatidylinositol-4,5-bisphosphate [PI(4,5)P 2 ]. Unexpectedly, the affinity of MA for tRNA Lys3 ( K d = 0.63 ± 0.03 μM) is approximately 1 order of magnitude greater than its affinity for PI(4,5)P 2 -enriched liposomes ( K d(apparent) = 10.2 ± 2.1 μM), and NMR studies indicate that tRNA Lys3 binding blocks MA association with liposomes, including those enriched with PI(4,5)P 2 , phosphatidylserine, and cholesterol. However, the affinity of MA for tRNA Lys3 is diminished by mutations or sample conditions that promote myristate exposure. Since Gag–Gag interactions are known to promote myristate exposure, our findings support virus assembly models in which membrane targeting and genome binding are mechanistically coupled. [ABSTRACT FROM AUTHOR]

The mature HIV-1 reverse transcriptase is a heterodimer that comprises 66 kDa (p66) and 51 kDa (p51) subunits. The latter is formed by HIV-1 protease-catalyzed removal of a C-terminal ribonuclease H domain from a p66 subunit. This proteolytic processing is a critical step in virus maturation and essential for viral infectivity. Here, we report that tRNA significantly enhances in vitro processing even at a substoichiometric tRNA:p66/p66 ratio. Other double-stranded RNAs have considerably less pronounced effect. Our data support a model where interaction of p66/p66 with tRNA introduces conformational asymmetry in the two subunits, permitting specific proteolytic processing of one p66 to provide the mature RT p66/p51 heterodimer. [ABSTRACT FROM AUTHOR]

Aminoacyl-tRNA synthetases are essential components in protein biosynthesis. Arginyl-tRNA synthetase (ArgRS) belongs to the small group of aminoacyl-tRNA synthetases requiring cognate tRNA for amino acid activation. The crystal structure of Escherichia coli ( Eco ) ArgRS has been solved in complex with tRNA Arg at 3.0-Å resolution. With this first bacterial tRNA complex, we are attempting to bridge the gap existing in structure–function understanding in prokaryotic tRNA Arg recognition. The structure shows a tight binding of tRNA on the synthetase through the identity determinant A20 from the D-loop, a tRNA recognition snapshot never elucidated structurally. This interaction of A20 involves 5 amino acids from the synthetase. Additional contacts via U20a and U16 from the D-loop reinforce the interaction. The importance of D-loop recognition in Eco ArgRS functioning is supported by a mutagenesis analysis of critical amino acids that anchor tRNA Arg on the synthetase; in particular, mutations at amino acids interacting with A20 affect binding affinity to the tRNA and specificity of arginylation. Altogether the structural and functional data indicate that the unprecedented ArgRS crystal structure represents a snapshot during functioning and suggest that the recognition of the D-loop by ArgRS is an important trigger that anchors tRNA Arg on the synthetase. In this process, A20 plays a major role, together with prominent conformational changes in several ArgRS domains that may eventually lead to the mature ArgRS:tRNA complex and the arginine activation. Functional implications that could be idiosyncratic to the arginine identity of bacterial ArgRSs are discussed. [ABSTRACT FROM AUTHOR]

Recent advances in transcriptome sequencing and analysis have revealed the complexity of the human genome. The majority (≈ 98%) of cellular transcripts is not translated into proteins and represents a vast, unchartered world of functional non-coding RNAs. Most of them adopt a well-defined three-dimensional structure to achieve their biological functions. However, only very few RNA structures are currently available which reflects the challenges associated with RNA crystallization. Nevertheless, these structures would represent a critical step in understanding functions of non-coding RNAs and their molecular mechanisms in the cell. The overall goal of this study is to develop an innovative and versatile tool to facilitate the functional study and crystallization of structured RNAs (stRNAs). In this work, we have engineered an antibody fragment from camelid heavy-chain antibody (nanobody) able to specifically bind with low nanomolar affinity to stRNA, while no binding could be detected for single-stranded DNA/RNA, double-stranded DNA/RNA or a negatively charged protein. However, this nanobody recognizes different and non-related stRNAs, this observation suggests that it binds to an epitope shared by these stRNAs. Finally, our data also show that the binding of the nanobody does not alter the secondary structure content of the stRNA as well as its unfolding/refolding processes during heat treatment. This work constitutes a successful proof of concept demonstrating that nanobodies can be engineered to recognize RNA-related epitopes. [ABSTRACT FROM AUTHOR]

Cellular growth crucially depends on protein synthesis and the abundance of translational components. Among them, aminoacyl-tRNAs play a central role in biosynthesis and shape the kinetics of mRNA translation, thus influencing protein production. Here, we used microarray-based approaches to determine the charging levels and tRNA abundance of Bacillus licheniformis . We observed an interesting cross-talk among tRNA expression, charging pattern, and growth rate. For a large subset of tRNAs, we found a co-regulated and augmented expression at high growth rate. Their tRNA aminoacylation level is kept relatively constant through riboswitch-regulated expression of the cognate aminoacyl-tRNA-synthetase (AARS). We show that AARSs with putative riboswitch-controlled expression are those charging tRNAs with amino acids which disfavor cell growth when individually added to the nutrient medium. Our results suggest that the riboswitch-regulated AARS expression in B. licheniformis is a powerful mechanism not only to maintain a constant ratio of aminoacyl-tRNA independent of the growth rate but concomitantly to control the intracellular level of free amino acids. [ABSTRACT FROM AUTHOR]

Protein synthesis is a key process in all living organisms. In eukaryotes, initiation factor 2 (eIF2) plays an important role in translation initiation as it selects and delivers the initiator tRNA to the small ribosomal subunit. Under stress conditions, phosphorylation of the α-subunit of eIF2 downregulates cellular protein synthesis. However, translation of certain mRNAs continues via the eIF2D-dependent non-canonical initiation pathway. The molecular mechanism of this process remains elusive. In addition, eIF2D plays a role in translation re-initiation and ribosome recycling. Currently, there has been no structural information of eIF2D. We have now determined the crystal structure of the C-terminal domains of eIF2D at 1.4-Å resolution. One domain has the fold similar to that of eIF1, which is crucial for the scanning and initiation codon selection. The second domain has a known SWIB/MDM2 fold, which was not observed before in other translation initiation factors. Our structure reveals atomic details of inter-domain interactions in the C-terminal part of eIF2D and sheds light on the possible role of these domains in eIF2D during translation. [ABSTRACT FROM AUTHOR]

[Display omitted] • Two independent smFRET assays reveal spontaneous intersubunit rotation of the ribosome. • Fraction of spontaneously fluctuating ribosomes depends on experimental conditions. • Thermally-driven ribosome rearrangements underlie translocation along mRNA. Mediated by elongation factor G (EF-G), ribosome translocation along mRNA is accompanied by rotational movement between ribosomal subunits. Here, we reassess whether the intersubunit rotation requires GTP hydrolysis by EF-G or can occur spontaneously. To that end, we employ two independent FRET assays, which are based on labeling either ribosomal proteins (bS6 and bL9) or rRNAs (h44 of 16S and H101 of 23S rRNA). Both FRET pairs reveal three FRET states, corresponding to the non-rotated, rotated and semi-rotated conformations of the ribosome. Both FRET assays show that in the absence of EF-G, pre-translocation ribosomes containing deacylated P-site tRNA undergo spontaneous intersubunit rotations between non-rotated and rotated conformations. While the two FRET pairs exhibit largely similar behavior, they substantially differ in the fraction of ribosomes showing spontaneous fluctuations. Nevertheless, instead of being an invariable intrinsic property of each FRET pair, the fraction of spontaneously fluctuating molecules changes in both FRET assays depending on experimental conditions. Our results underscore importance of using multiple FRET pairs in studies of ribosome dynamics and highlight the role of thermally-driven large-scale ribosome rearrangements in translation. [ABSTRACT FROM AUTHOR]

[Display omitted] • The T-loop motif frequently mediates tertiary interactions in RNA. • Activity-based screen used to explore T-loop mediated interactions in riboswitches. • Results from the screen were consistent with phylogenetic analysis. • Consensus sequence for three different T-loop receptor motifs. • Results show T-loop/T-loop receptor interactions are context dependent. The architecture and folding of complex RNAs is governed by a limited set of highly recurrent structural motifs that form long-range tertiary interactions. One of these motifs is the T-loop, which was first identified in tRNA but is broadly distributed across biological RNAs. While the T-loop has been examined in detail in different biological contexts, the various receptors that it interacts with are not as well defined. In this study, we use a cell-based genetic screen in concert with bioinformatic analysis to examine three different, but related, T-loop receptor motifs found in the flavin mononucleotide (FMN) and cobalamin (Cbl) riboswitches. As a host for different T-loop receptors, we employed the env 8 class-II Cbl riboswitch, an RNA that uses two T-loop motifs for both folding and supporting the ligand binding pocket. A set of libraries was created in which select nucleotides that participate in the T-loop/T-loop receptor (TL/TLR) interaction were fully randomized. Library members were screened for their ability to support Cbl-dependent expression of a reporter gene. While T-loops appear to be variable in sequence, we find that the functional sequence space is more restricted in the Cbl riboswitch, suggesting that TL/TLR interactions are context dependent. Our data reveal clear sequence signatures for the different types of receptor motifs that align with phylogenic analysis of these motifs in the FMN and Cbl riboswitches. Finally, our data suggest the functional contribution of various nucleobase-mediated long-range interactions within the riboswitch subclass of TL/TLR interactions that are distinct from those found in other RNAs. [ABSTRACT FROM AUTHOR]

Ribonuclease P (RNase P) is the enzyme that endonucleolytically removes 5′-precursor sequences from tRNA transcripts in all domains of life. RNase P activities are either ribonucleoprotein (RNP) or protein-only RNase P (PRORP) enzymes, raising the question about the mechanistic strategies utilized by these architecturally different enzyme classes to catalyze the same type of reaction. Here, we analyzed the kinetics and cleavage-site selection by PRORP3 from Arabidopsis thaliana ( At PRORP3) using precursor tRNAs (pre-tRNAs) with individual modifications at the canonical cleavage site, with either R p- or S p-phosphorothioate, or 2′-deoxy, 2′-fluoro, 2′-amino, or 2′-O-methyl substitutions. We observed a small but robust rescue effect of S p-phosphorothioate-modified pre-tRNA in the presence of thiophilic Cd 2 + ions, consistent with metal-ion coordination to the (pro-) S p-oxygen during catalysis. S p-phosphorothioate, 2′-deoxy, 2′-amino, and 2′- O -methyl modification redirected the cleavage mainly to the next unmodified phosphodiester in the 5′-direction. Our findings are in line with the 2′-OH substituent at nucleotide − 1 being involved in an H-bonding acceptor function. In contrast to bacterial RNase P, At PRORP3 was found to be able to utilize the canonical and upstream cleavage site with similar efficiency (corresponding to reduced cleavage fidelity), and the two cleavage pathways appear less interdependent than in the bacterial RNA-based system. [ABSTRACT FROM AUTHOR]

It is general wisdom that termination of bacterial protein synthesis is obligatorily followed by recycling governed by the factors ribosomal recycling factor (RRF), EF-G, and IF3, where the ribosome dissociates into its subunits. In contrast, a recently described 70S-scanning mode of initiation holds that after termination, scanning of 70S can be triggered by fMet-tRNA to the initiation site of a downstream cistron. Here, we analyze the apparent conflict. We constructed a bicistronic mRNA coding for luciferases and showed with a highly resolved in vitro system that the expression of the second cistron did not at all depend on the presence of active RRF. An in vivo analysis cannot be performed in a straightforward way, since RRF is essential for viability and therefore, the RRF gene cannot be knocked out. However, we found an experimental window, where the RRF amount could be reduced to below 2.5%, and in this situation, the expression of the second cistron of a bicistronic luciferase mRNA was only moderately reduced. Both in vitro and in vivo results suggested that RRF-dependent recycling is not an obligatory step after termination, in agreement with the previous findings concerning 70S-scanning initiation. In this view, recycling after termination is a special case of the general RRF function, which happens whenever fMet-tRNA is not available for triggering 70S scanning. [ABSTRACT FROM AUTHOR]

Elongation factor G (EF-G) is a universally conserved translational GTPase that promotes the translocation of tRNA and mRNA through the ribosome. EF-G binds to the ribosome in a GTP-bound form and subsequently catalyzes GTP hydrolysis. The contribution of the ribosome-stimulated GTP hydrolysis by EF-G to tRNA/mRNA translocation remains debated. Here, we show that while EF-G•GDP does not stably bind to the ribosome and induce translocation, EF-G•GDP in complex with phosphate group analogs BeF 3 − and AlF 4 − promotes the translocation of tRNA and mRNA. Furthermore, the rates of mRNA translocation induced by EF-G in the presence of GTP and a non-hydrolyzable analog of GTP, GDP•BeF 3 − are similar. Our results are consistent with the model suggesting that GTP hydrolysis is not directly coupled to mRNA/tRNA translocation. Hence, GTP binding is required to induce the activated, translocation-competent conformation of EF-G while GTP hydrolysis triggers EF-G release from the ribosome. [ABSTRACT FROM AUTHOR]

N6-methyladenosine (m 6 A) is ubiquitously present in the RNA of living organisms from Escherichia coli to humans. Methyltransferases that catalyze adenosine methylation are drastically different in specificity from modification of single residues in bacterial ribosomal or transfer RNA to modification of thousands of residues spread among eukaryotic mRNA. Interactions that are formed by m 6 A residues range from RNA–RNA tertiary contacts to RNA–protein recognition. Consequences of the modification loss might vary from nearly negligible to complete reprogramming of regulatory pathways and lethality. In this review, we summarized current knowledge on enzymes that introduce m 6 A modification, ways to detect m 6 A presence in RNA and the functional role of this modification everywhere it is present, from bacteria to humans. [ABSTRACT FROM AUTHOR]

The ribosome is a large two-subunit ribonucleoprotein machine that translates the genetic code in all cells, synthesizing proteins according to the sequence of the mRNA template. During translation, the primary substrates, transfer RNAs, pass through binding sites formed between the two subunits. Multiple interactions between the ribosomal subunits, termed intersubunit bridges, keep the ribosome intact and at the same time govern dynamics that facilitate the various steps of translation such as transfer RNA–mRNA movement. Here, we review the molecular nature of these intersubunit bridges, how they change conformation during translation, and their functional roles in the process. [ABSTRACT FROM AUTHOR]

[Display omitted] • Detection of amyloids in cells requires unique spectral signatures. • The aryl alkyne F CC is a site-specific conformational probe of α-syn aggregation. • Cellular Raman mapping uniquely identifies internalized F CC -labeled α-syn fibrils. • Spectral analysis reveals differences in lipid and protein recruitment to fibrils. • Raman spectral imaging is a powerful approach to study amyloid structure in cells. α-Synuclein (α-syn) is an intrinsically disordered protein involved in a group of diseases collectively termed synucleinopathies, characterized by the aggregation of α-syn to form insoluble, β-sheet-rich amyloid fibrils. Amyloid fibrils are thought to contribute to disease progression through cell-to-cell transmission, templating and propagating intracellular amyloid formation. Raman spectral imaging offers a direct characterization of protein secondary structure via the amide-I backbone vibration; however, specific detection of α-syn conformational changes against the background of other cellular components presents a challenge. Here, we demonstrate the ability to unambiguously identify cellularly internalized α-syn fibrils by coupling Raman spectral imaging with the use of a genetically encoded aryl alkyne, 4-ethynyl- l -phenylalanine (F CC), through amber codon suppression. The alkyne stretch (CC) of F CC provides a spectrally unique molecular vibration without interference from native biomolecules. Cellular uptake of F CC -α-syn fibrils formed in vitro was visualized in cultured human SH-SY5Y neuroblastoma cells by Raman spectral imaging. Fibrils appear as discrete cytosolic clusters of varying sizes, found often at the cellular periphery. Raman spectra of internalized fibrils exhibit frequency shifts and spectral narrowing relative to in vitro fibrils, highlighting the environmental sensitivity of the alkyne vibration. Interestingly, spectral analysis reveals variations in lipid and protein recruitment to these aggregates, and in some cases, secondary structural changes in the fibrils are observed. This work sets the groundwork for future Raman spectroscopic investigations using a similar approach of an evolved aminoacyl-tRNA synthetase/tRNA pair to incorporate F CC into endogenous amyloidogenic proteins to monitor their aggregation in cells. [ABSTRACT FROM AUTHOR]

Withstanding 3.5 billion years of genetic drift, the canonical genetic code remains such a fundamental foundation for the complexity of life that it is highly conserved across all three phylogenetic domains. Genome engineering technologies are now making it possible to rationally change the genetic code, offering resistance to viruses, genetic isolation from horizontal gene transfer, and prevention of environmental escape by genetically modified organisms. We discuss the biochemical, genetic, and technological challenges that must be overcome in order to engineer the genetic code. [ABSTRACT FROM AUTHOR]

Synthesis of asparaginyl-tRNA (Asn-tRNA Asn ) in bacteria can be formed either by directly ligating Asn to tRNA Asn using an asparaginyl-tRNA synthetase (AsnRS) or by synthesizing Asn on the tRNA. In the latter two-step indirect pathway, a non-discriminating aspartyl-tRNA synthetase (ND-AspRS) attaches Asp to tRNA Asn and the amidotransferase GatCAB transamidates the Asp to Asn on the tRNA. GatCAB can be similarly used for Gln-tRNA Gln formation. Most bacteria are predicted to use only one route for Asn-tRNA Asn formation. Given that Bacillus halodurans and Bacillus subtilis encode AsnRS for Asn-tRNA Asn formation and Asn synthetases to synthesize Asn and GatCAB for Gln-tRNA Gln synthesis, their AspRS enzymes were thought to be specific for tRNA Asp . However, we demonstrate that the AspRSs are non-discriminating and can be used with GatCAB to synthesize Asn. The results explain why B . subtilis with its Asn synthetase genes knocked out is still an Asn prototroph. Our phylogenetic analysis suggests that this may be common among Firmicutes and 30% of all bacteria. In addition, the phylogeny revealed that discrimination toward tRNA Asp by AspRS has evolved independently multiple times. The retention of the indirect pathway in B . subtilis and B . halodurans likely reflects the ancient link between Asn biosynthesis and its use in translation that enabled Asn to be added to the genetic code. [ABSTRACT FROM AUTHOR]

Protein-only RNase Ps (PRORPs) are a recently discovered class of RNA processing enzymes that catalyze maturation of the 5' end of precursor tRNAs in Eukaryotes. PRORPs are found in the nucleus and/or organelles of most eukaryotic organisms. Arabidopsis thaliana is a representative organism that contains PRORP enzymes (PRORP1, PRORP2 and PRORP3) in both its nucleus and its organelles; PRORP2 and PRORP3 localize to the nucleus and PRORP1 localizes to the chloroplast and the mitochondria. Apart from their identification, almost nothing is known about the structure and function of PRORPs that act in the nucleus. Here, we use a combination of biochemical assays and X-ray crystallography to characterize A. thaliana PRORP2. We solved the crystal structure of PRORP2 (3.2 Å) revealing an overall V-shaped protein and conserved metallonuclease active-site structure. Our biochemical studies indicate that PRORP2 requires Mg2+ for catalysis and catalyzes the maturation of nuclear encoded substrates up to 10-fold faster than mitochondrial encoded precursor nad6 t-element under single-turnover conditions. We also demonstrate that PRORP2 preferentially binds precursor tRNAs containing short 5' leaders and 3' trailers; however, leader and trailer lengths do not significantly alter the observed rate constants of PRORP2 in single-turnover cleavage assays. Our data provide a biochemical and structural framework to begin understanding how nuclear localized PRORPs recognize and cleave their substrates. [ABSTRACT FROM AUTHOR]

Human tRNA 3 Lys is the primer for reverse transcription of HIV; the 3′ end is complementary to the primer-binding site on HIV RNA. The complementarity ends at the 18th base, A58, which in tRNA 3 Lys is modified to remove Watson–Crick pairing. Motivated to test the role of the modification in terminating the primer-binding sequence and thus limiting run-on transcription, we asked how the modification of RNA could be accomplished. tRNA m 1 A58 methyltransferase (m 1 A58 MTase) methylates N1 of A58, which is buried in the TΨC-loop of tRNA, from cofactor S -adenosyl- l -methionine. This conserved tRNA modification is essential for stability of initiator tRNA in Saccharomyces cerevisiae . Reported here, three structures of human tRNA m 1 A58 MTase in complex with human tRNA 3 Lys and the product S -adenosyl- l -homocysteine show a dimer of heterodimers in which each heterodimer comprises a catalytic chain, Trm61, and a homologous but noncatalytic chain, Trm6, repurposed as a tRNA-binding subunit that acts in trans ; tRNAs bind across the dimer interface such that Trm6 from the opposing heterodimer brings A58 into the active site of Trm61. T-loop and D-loop are splayed apart showing how A58, normally buried in tRNA, becomes accessible for modification. This result has broad impact on our understanding of the mechanisms of modifying internal sites in folded tRNA. The structures serve as templates for design of inhibitors that could be used to test tRNA m 1 A58 MTase's impact on retroviral priming and transcription. [ABSTRACT FROM AUTHOR]

Escherichia coli TcdA (also known as CsdL) was previously shown to catalyze the ATP-dependent dehydration/cyclization of hypermodified tRNA N 6 -threonylcarbamoyladenosine into further cyclic N 6 -threonylcarbamoyladenosine. In this study, we report the X-ray crystal structures of E . coli TcdA with either AMP or ATP bound. The AMP/ATP-bound N-terminal sub-domain of TcdA resembles the ATP-binding Rossmann fold of E . coli ThiF and MoeB that are enzymes respectively taking part in the biosynthesis of thiamine and molybdopterin; however, the remaining C-terminal sub-domain of TcdA adopts a structure unrelated to any other known folds. In TcdA, the ATP-utilizing adenylation of tRNA N 6 -threonylcarbamoyladenosine and a subsequent thioester formation via an active cysteine, similar to the mechanisms in ThiF and MoeB, could take place for the dehydratase function. Analysis of the structure with sequence alignment suggests the disordered Cys234 of TcdA as the most likely catalytic residue. The structure further indicates that the C-terminal sub-domain can provide a binding interface for the tRNA substrate. Binding study using the surface mutants of TcdA and tRNA reveals that the positively charged regions of mainly the C-terminal sub-domain are important for the tRNA recognition. [ABSTRACT FROM AUTHOR]

Adenosine triphosphate (ATP) is the energy currency of living cells. Even though ATP powers virtually all energy-dependent activities, most cellular ATP is utilized in protein synthesis via tRNA aminoacylation and guanosine triphosphate regeneration. Magnesium (Mg 2 + ), the most common divalent cation in living cells, plays crucial roles in protein synthesis by maintaining the structure of ribosomes, participating in the biochemistry of translation initiation and functioning as a counterion for ATP. A non-physiological increase in ATP levels hinders growth in cells experiencing Mg 2 + limitation because ATP is the most abundant nucleotide triphosphate in the cell, and Mg 2 + is also required for the stabilization of the cytoplasmic membrane and as a cofactor for essential enzymes. We propose that organisms cope with Mg 2 + limitation by decreasing ATP levels and ribosome production, thereby reallocating Mg 2 + to indispensable cellular processes. [ABSTRACT FROM AUTHOR]

We have studied the pH dependence of the rate of termination of bacterial protein synthesis catalyzed by a class-1 release factor (RF1 or RF2). We used a classical quench-flow technique and a newly developed stopped-flow technique that relies on the use of fluorescently labeled peptides. We found the termination rate to increase with increasing pH and, eventually, to saturate at about 70 s − 1 with an apparent p K a value of about 7.6. From our data, we suggest that class-1 RF termination is rate limited by the chemistry of ester bond hydrolysis at low pH and by a stop-codon-dependent and pH-independent conformational change of RFs at high pH. We propose that RF-dependent termination depends on the participation of a hydroxide ion rather than a water molecule in the hydrolysis of the ester bond between the P-site tRNA and its peptide chain. We provide a simple explanation for why the rate of termination saturated at high pH in our experiments but not in those of others. [ABSTRACT FROM AUTHOR]

Joining of the large, 50S, ribosomal subunit to the small, 30S, ribosomal subunit initiation complex (IC) during bacterial translation initiation is catalyzed by the initiation factor (IF) IF2. Because the rate of subunit joining is coupled to the IF, transfer RNA (tRNA), and mRNA codon compositions of the 30S IC, the subunit joining reaction functions as a kinetic checkpoint that regulates the fidelity of translation initiation. Recent structural studies suggest that the conformational dynamics of the IF2·tRNA sub-complex forming on the intersubunit surface of the 30S IC may play a significant role in the mechanisms that couple the rate of subunit joining to the IF, tRNA, and codon compositions of the 30S IC. To test this hypothesis, we have developed a single-molecule fluorescence resonance energy transfer signal between IF2 and tRNA that has enabled us to monitor the conformational dynamics of the IF2·tRNA sub-complex across a series of 30S ICs. Our results demonstrate that 30S ICs undergoing rapid subunit joining display a high affinity for IF2 and an IF2·tRNA sub-complex that primarily samples a single conformation. In contrast, 30S ICs that undergo slower subunit joining exhibit a decreased affinity for IF2 and/or a change in the conformational dynamics of the IF2·tRNA sub-complex. These results strongly suggest that 30S IC-driven changes in the stability of IF2 and the conformational dynamics of the IF2·tRNA sub-complex regulate the efficiency and fidelity of subunit joining during translation initiation. [ABSTRACT FROM AUTHOR]

[Display omitted] • The first structure of Saccharomyces cerevisiae ATE1 (Sc ATE1) has been determined. • Cryo-EM and SAXS data corroborate crystalline and solution behavior of Sc ATE1. • Apo Sc ATE1 contrasts with the structure of Zn2+-bound Kluyveromyces lactis ATE1. • Based on structural data, a mechanism of N-terminal arginylation has been proposed. Eukaryotic post-translational arginylation, mediated by the family of enzymes known as the arginyltransferases (ATE1s), is an important post-translational modification that can alter protein function and even dictate cellular protein half-life. Multiple major biological pathways are linked to the fidelity of this process, including neural and cardiovascular developments, cell division, and even the stress response. Despite this significance, the structural, mechanistic, and regulatory mechanisms that govern ATE1 function remain enigmatic. To that end, we have used X-ray crystallography to solve the crystal structure of ATE1 from the model organism Saccharomyces cerevisiae ATE1 (Sc ATE1) in the apo form. The three-dimensional structure of Sc ATE1 reveals a bilobed protein containing a GCN5-related N-acetyltransferase (GNAT) fold, and this crystalline behavior is faithfully recapitulated in solution based on size-exclusion chromatography-coupled small angle X-ray scattering (SEC-SAXS) analyses and cryo-EM 2D class averaging. Structural superpositions and electrostatic analyses point to this domain and its domain-domain interface as the location of catalytic activity and tRNA binding, and these comparisons strongly suggest a mechanism for post-translational arginylation. Additionally, our structure reveals that the N-terminal domain, which we have previously shown to bind a regulatory [Fe-S] cluster, is dynamic and disordered in the absence of metal bound in this location, hinting at the regulatory influence of this region. When taken together, these insights bring us closer to answering pressing questions regarding the molecular-level mechanism of eukaryotic post-translational arginylation. [ABSTRACT FROM AUTHOR]

Ribosomes control the missense error rate of ~ 10 − 4 during translation though quantitative contributions of individual mechanistic steps of the conformational changes yet to be fully determined. Biochemical and biophysical studies led to a qualitative tRNA selection model in which ribosomal A-site residues A1492 and A1493 (A1492/3) flip out in response to cognate tRNA binding, promoting the subsequent reactions, but not in the case of near-cognate or non-cognate tRNA. However, this model was recently questioned by X-ray structures revealing conformations of extrahelical A1492/3 and domain closure of the decoding center in both cognate and near-cognate tRNA bound ribosome complexes, suggesting that the non-specific flipping of A1492/3 has no active role in tRNA selection. We explore this question by carrying out molecular dynamics simulations, aided with fluorescence and NMR experiments, to probe the free energy cost of extrahelical flipping of 1492/3 and the strain energy associated with domain conformational change. Our rigorous calculations demonstrate that the A1492/3 flipping is indeed a specific response to the binding of cognate tRNA, contributing 3 kcal/mol to the specificity of tRNA selection. Furthermore, the different A-minor interactions in cognate and near-cognate complexes propagate into the conformational strain and contribute another 4 kcal/mol in domain closure. The recent structure of ribosome with features of extrahelical A1492/3 and closed domain in near-cognate complex is reconciled by possible tautomerization of the wobble base pair in mRNA–tRNA. These results quantitatively rationalize other independent experimental observations and explain the ribosomal discrimination mechanism of selecting cognate versus near-cognate tRNA. [ABSTRACT FROM AUTHOR]

Abstract: The 21st amino acid, selenocysteine (Sec), is incorporated translationally into proteins and is synthesized on its specific tRNA (tRNASec). In Bacteria, the selenocysteine synthase SelA converts Ser-tRNASec, formed by seryl-tRNA synthetase, to Sec-tRNASec. SelA, a member of the fold-type-I pyridoxal 5′-phosphate-dependent enzyme superfamily, has an exceptional homodecameric quaternary structure with a molecular mass of about 500kDa. Our previously determined crystal structures of Aquifex aeolicus SelA complexed with tRNASec revealed that the ring-shaped decamer is composed of pentamerized SelA dimers, with two SelA dimers arranged to collaboratively interact with one Ser-tRNASec. The SelA catalytic site is close to the dimer–dimer interface, but the significance of the dimer pentamerization in the catalytic site formation remained elusive. In the present study, we examined the quaternary interactions and demonstrated their importance for SelA activity by systematic mutagenesis. Furthermore, we determined the crystal structures of “depentamerized” SelA variants with mutations at the dimer–dimer interface that prevent pentamerization. These dimeric SelA variants formed a distorted and inactivated catalytic site and confirmed that the pentamer interactions are essential for productive catalytic site formation. Intriguingly, the conformation of the non-functional active site of dimeric SelA shares structural features with other fold-type-I pyridoxal 5′-phosphate-dependent enzymes with native dimer or tetramer (dimer-of-dimers) quaternary structures. [Copyright &y& Elsevier]