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A model Post-Termination Complex (PoTC) used for the discovery of Ribosome Recycling Factor (RRF) was purified and characterized by cryo-electron microscopic analysis and biochemical methods. We established that the model PoTC has mostly one tRNA, at the P/E or P/P position, together with one mRNA. The structural studies were supported by the biochemical measurement of bound tRNA and mRNA. Using this substrate, we establish that the release of tRNA, release of mRNA and splitting of ribosomal subunits occur during the recycling reaction. Order of these events is tRNA release first followed by mRNA release and splitting almost simultaneously. Moreover, we demonstrate that IF3 is not involved in any of the recycling reactions but simply prevents the re-association of split ribosomal subunits. Our finding demonstrates that the important function of RRF includes the release of mRNA, which is often missed by the use of a short ORF with the Shine-Dalgarno sequence near the termination site. [ABSTRACT FROM AUTHOR]

Small protein B, SmpB, is a tmRNA-specific binding protein essential for trans-translation. We examined the interaction between SmpB and tmRNA from Thermus thermophilus, using biochemical and NMR methods. Chemical footprinting analyses using full-length tmRNA demonstrated that the sites protected upon SmpB binding are located exclusively in the tRNA-like domain (TLD) of tmRNA. To clarify the SmpB binding sites, we constructed several segments derived from TLD. Optical biosensor interaction analyses and melting profile analyses with mutational studies showed that SmpB efficiently binds to only a 30-nt segment that forms a stem and loop, with the 5′ and 3′ extensions composed of the D-loop and variable-loop analogues. The conserved sequences, 16UCGA and 319GAC, in the extensions are responsible for the SmpB binding. These results agree with the those visualized by the cocrystal structure of TLD and SmpB from Aquifex aeolicus. In addition, NMR chemical shift mapping analyses, using the 30-nt segment and 15N-labeled SmpB, revealed the characteristic RNA binding mode. The hydrogen bond pattern around β2 changes, with the Gly in β2, which acts as a hinge, showing the largest chemical shift change. It appears that SmpB undergoes structural changes indicating an induced fit upon binding to the specific region of TLD. [ABSTRACT FROM PUBLISHER]

Cell-free protein synthesis is useful for synthesizing difficult targets. The site-specific incorporation of non-natural amino acids into proteins is a powerful protein engineering method. In this study, we optimized the protocol for cell extract preparation from the Escherichia coli strain RFzero-iy, which is engineered to lack release factor 1 (RF-1). The BL21(DE3)-based RFzero-iy strain exhibited quite high cell-free protein productivity, and thus we established the protocols for its cell culture and extract preparation. In the presence of 3-iodo-l-tyrosine (IY), cell-free protein synthesis using the RFzero-iy-based S30 extract translated the UAG codon to IY at various sites with a high translation efficiency of >90%. In the absence of IY, the RFzero-iy-based cell-free system did not translate UAG to any amino acid, leaving UAG unassigned. Actually, UAG was readily reassigned to various non-natural amino acids, by supplementing them with their specific aminoacyl-tRNA synthetase variants (and their specific tRNAs) into the system. The high incorporation rate of our RFzero-iy-based cell-free system enables the incorporation of a variety of non-natural amino acids into multiple sites of proteins. The present strategy to create the RFzero strain is rapid, and thus promising for RF-1 deletions of various E. coli strains genomically engineered for specific requirements. [ABSTRACT FROM AUTHOR]

In the final step of tRNA splicing, the 2′-phosphotransferase catalyzes the transfer of the extra 2′-phosphate from the precursor-ligated tRNA to NAD. We have determined the crystal structure of the 2′-phosphotransferase protein from Aeropyrum pernix K1 at 2.8Å resolution. The structure of the 2′-phosphotransferase contains two globular domains (N and C-domains), which form a cleft in the center. The N-domain has the winged helix motif, a subfamily of the helix-turn-helix family, which is shared by many DNA-binding proteins. The C-domain of the 2′-phosphotransferase superimposes well on the NAD-binding fold of bacterial (diphtheria) toxins, which catalyze the transfer of ADP ribose from NAD to target proteins, indicating that the mode of NAD binding by the 2′-phosphotransferase could be similar to that of the bacterial toxins. The conserved basic residues are assembled at the periphery of the cleft and could participate in the enzyme contact with the sugar-phosphate backbones of tRNA. The modes by which the two functional domains recognize the two different substrates are clarified by the present crystal structure of the 2′-phosphotransferase. [Copyright &y& Elsevier]

Transfer RNA (tRNA) modifications are critical for protein synthesis. Queuosine (Q), a 7-deaza-guanosine derivative, is present in tRNA anticodons. In vertebrate tRNAs for Tyr and Asp, Q is further glycosylated with galactose and mannose to generate galQ and manQ, respectively. However, biogenesis and physiological relevance of Q-glycosylation remain poorly understood. Here, we biochemically identified two RNA glycosylases, QTGAL and QTMAN, and successfully reconstituted Q-glycosylation of tRNAs using nucleotide diphosphate sugars. Ribosome profiling of knockout cells revealed that Q-glycosylation slowed down elongation at cognate codons, UAC and GAC (GAU), respectively. We also found that galactosylation of Q suppresses stop codon readthrough. Moreover, protein aggregates increased in cells lacking Q-glycosylation, indicating that Q-glycosylation contributes to proteostasis. Cryo-EM of human ribosome-tRNA complex revealed the molecular basis of codon recognition regulated by Q-glycosylations. Furthermore, zebrafish qtgal and qtman knockout lines displayed shortened body length, implying that Q-glycosylation is required for post-embryonic growth in vertebrates. [Display omitted] • Two tRNA Q-glycosylation enzymes QTGAL and QTMAN were identified • Q-glycosylation of tRNA contributes to optimal translation and proteostasis • Structural analyses revealed molecular basis of codon recognition by Q-glycosylation • Q-glycosylation of tRNA is required for post-embryonic growth of zebrafish Queosine (Q) is a modified nucleoside present in tRNA anticodons. Identification and characterization of two vertebrate Q-glycosylases unveil new regulatory mechanism in protein translation and proteostasis. [ABSTRACT FROM AUTHOR]

A core event in the integrated stress response, an adaptive pathway common to all eukaryotic cells in response to various stress stimuli, is the phosphorylation of eukaryotic translation initiation factor 2 (eIF2). Normally, unphosphorylated eIF2 transfers the methionylated initiator tRNA to the ribosome in a guanosine 5′-triphosphate–dependent manner. By contrast, phosphorylated eIF2 inhibits its specific guanine nucleotide exchange factor, eIF2B. T elucidate how the eIF2 phosphorylation status regulates the eIF2B activity, we determined cryo–electron microscopic and crystallographic structures of eIF2B in complex with unphosphorylated or phosphorylated eIF2. The unphosphorylated and phosphorylated forms of eIF2 bind to eIF2B in completely different manners: the nucleotide exchange-active and -inactive modes, respectively. These structures explain how phosphorylated eIF2 dominantly inhibits the nucleotide exchange activity of eIF2B. [ABSTRACT FROM AUTHOR]

Abstract: A phylogenetically conserved ribosomal protein L16p/L10e organizes the architecture of the aminoacyl tRNA binding site on the large ribosomal subunit. Eukaryotic L10 also exhibits a variety of cellular activities, and, in particular, human L10 is known as a putative tumor suppressor, QM. We have determined the 2.5-Å crystal structure of the human L10 core domain that corresponds to residues 34–182 of the full-length 214 amino acids. Its two-layered α+β architecture is significantly similar to those of the archaeal and bacterial homologues, substantiating a high degree of structural conservation across the three phylogenetic domains. A cation-binding pocket formed between α2 and β6 is similar to that of the archaeal L10 protein but appears to be better ordered. Previously reported L10 mutations that cause defects in the yeast ribosome are clustered around this pocket, indicating that its integrity is crucial for its role in L10 function. Characteristic interactions among Arg90–Trp171–Arg139 guide the C-terminal part outside of the central fold, implying that the eukaryote-specific C-terminal extension localizes on the outer side of the ribosome. [Copyright &y& Elsevier]

Summary: Elongation factor G (EF-G) catalyzes tRNA translocation on the ribosome. Here a cryo-EM reconstruction of the 70S•EF-G ribosomal complex at 7.3 Å resolution and the crystal structure of EF-G-2•GTP, an EF-G homolog, at 2.2 Å resolution are presented. EF-G-2•GTP is structurally distinct from previous EF-G structures, and in the context of the cryo-EM structure, the conformational changes are associated with ribosome binding and activation of the GTP binding pocket. The P loop and switch II approach A2660-A2662 in helix 95 of the 23S rRNA, indicating an important role for these conserved bases. Furthermore, the ordering of the functionally important switch I and II regions, which interact with the bound GTP, is dependent on interactions with the ribosome in the ratcheted conformation. Therefore, a network of interaction with the ribosome establishes the active GTP conformation of EF-G and thus facilitates GTP hydrolysis and tRNA translocation. [Copyright &y& Elsevier]

Summary: In the initiation phase of bacterial translation, the 30S ribosomal subunit captures mRNA in preparation for binding with initiator tRNA. The purine-rich Shine-Dalgarno (SD) sequence, in the 5′ untranslated region of the mRNA, anchors the 30S subunit near the start codon, via base pairing with an anti-SD (aSD) sequence at the 3′ terminus of 16S rRNA. Here, we present the 3.3 Å crystal structure of the Thermus thermophilus 30S subunit bound with an mRNA mimic. The duplex formed by the SD and aSD sequences is snugly docked in a “chamber” between the head and platform domains, demonstrating how the 30S subunit captures and stabilizes the otherwise labile SD helix. This location of the SD helix is suitable for the placement of the start codon AUG in the immediate vicinity of the mRNA channel, in agreement with reported crosslinks between the second position of the start codon and G1530 of 16S rRNA. [Copyright &y& Elsevier]

Abstract: Asparaginyl-tRNA synthetase (AsnRS) is a member of the class-II aminoacyl-tRNA synthetases, and is responsible for catalyzing the specific aminoacylation of tRNAAsn with asparagine. Here, the crystal structure of AsnRS from Pyrococcus horikoshii, complexed with asparaginyl-adenylate (Asn-AMP), was determined at 1.45 Å resolution, and those of free AsnRS and AsnRS complexed with an Asn-AMP analog (Asn-SA) were solved at 1.98 and 1.80 Å resolutions, respectively. All of the crystal structures have many solvent molecules, which form a network of hydrogen-bonding interactions that surrounds the entire AsnRS molecule. In the AsnRS/Asn-AMP complex (or the AsnRS/Asn-SA), one side of the bound Asn-AMP (or Asn-SA) is completely covered by the solvent molecules, which complement the binding site. In particular, two of these water molecules were found to interact directly with the asparagine amide and carbonyl groups, respectively, and to contribute to the formation of a pocket highly complementary to the asparagine side-chain. Thus, these two water molecules appear to play a key role in the strict recognition of asparagine and the discrimination against aspartic acid by the AsnRS. This water-assisted asparagine recognition by the AsnRS strikingly contrasts with the fact that the aspartic acid recognition by the closely related aspartyl-tRNA synthetase is achieved exclusively through extensive interactions with protein amino acid residues. Furthermore, based on a docking model of AsnRS and tRNA, a single arginine residue (Arg83) in the AsnRS was postulated to be involved in the recognition of the third position of the tRNAAsn anticodon (U36). We performed a mutational analysis of this particular arginine residue, and confirmed its significance in the tRNA recognition. [Copyright &y& Elsevier]

2-Thioribothymidine (s²T) is a modified nucleoside of U, specifically found at position 54 of tRNAs from extreme thermophilic microorganisms. The function of the 2-thiocarbonyl group of s²T54 is thermostabilization of the three-dimensional structure of tRNA; however, its biosynthesis has not been clarified until now. Using an in vivo tRNA labeling experiment, we demonstrate that the sulfur atom of s²T in tRNA is derived from cysteine or sulfate. We attempted to reconstitute 2-thiolation of s2T in vitro, using a cell extract of Thermus thermophilus. Specific 2-thiolation of ribothymidine, at position 54, was observed in vitro, in the presence of ATP. Using this assay, we found a strong temperature dependence of the 2-thiolation reaction in vitro as well as expression of 2-thiolation enzymes in vivo. These results suggest that the variable content of s2T in vivo at different temperatures may be explained by the above characteristics of the enzymes responsible for the 2-thiolation reaction. Furthermore, we found that another posttranscriptionally modified nucleoside, 1-methyladenosine at position 58, is required for the efficient 2-thiolation of ribothymidine 54 both in vivo and in vitro. [ABSTRACT FROM AUTHOR]

Tyrosyl-tRNA synthetase (TyrRS) catalyzes the tyrosylation of tRNATyr in a two-step reaction. TyrRS has the “HIGH” and “KMSKS” motifs, which play essential roles in the formation of the tyrosyl-adenylate from tyrosine and ATP. Here, we determined the crystal structures of Archaeoglobus fulgidus and Pyrococcus horikoshii TyrRSs in the l-tyrosine-bound form at 1.8Å and 2.2Å resolutions, respectively, and that of Aeropyrum pernix TyrRS in the substrate-free form at 2.2 Å. The conformation of the KMSKS motif differs among the three TyrRSs. In the A.pernix TyrRS, the KMSKS loop conformation corresponds to the ATP-bound “closed” form. In contrast, the KMSKS loop of the P.horikoshii TyrRS forms a novel 310 helix, which appears to correspond to the “semi-closed” form. This conformation enlarges the entrance to the tyrosine-binding pocket, which facilitates the pyrophosphate ion release after the tyrosyl-adenylate formation, and probably is involved in the initial tRNA binding. The KMSSS loop of the A.fulgidus TyrRS is somewhat farther from the active site and is stabilized by hydrogen bonds. Based on the three structures, possible structural changes of the KMSKS motif during the tyrosine activation reaction are discussed. We suggest that the insertion sequence just before the KMSKS motif, which exists in some archaeal species, enhances the binding affinity of the TyrRS for its cognate tRNA. In addition, a non-proline cis peptide bond, which is involved in the tRNA binding, is conserved among the archaeal TyrRSs. [Copyright &y& Elsevier]

The bacterial tRNA adenosine deaminase (TadA) generates inosine by deaminating the adenosine residue at the wobble position of tRNAArg-2. This modification is essential for the decoding system. In this study, we determined the crystal structure of Aquifex aeolicus TadA at a 1.8-Å resolution. This is the first structure of a deaminase acting on tRNA. A. aeolicus TadA has an α/β/α three-layered fold and forms a homodimer. The A. aeolicus TadA dimeric structure is completely different from the tetrameric structure of yeast CDD1, which deaminates mRNA and cytidine, but is similar to the dimeric structure of yeast cytosine deaminase. However, in the A. aeolicus TadA structure, the shapes of the C-terminal helix and the regions between the β4 and β5 strands are quite distinct from those of yeast cytosine deaminase and a large cavity is produced. This cavity contains many conserved amino acid residues that are likely to be involved in either catalysis or tRNA binding. We made a docking model of TadA with the tRNA anticodon stem loop. [ABSTRACT FROM AUTHOR]