In many organisms, the UGA stop codon is recoded to insert selenocysteine (Sec) into proteins. Sec incorporation in bacteria is directed by an mRNA element, known as the Sec-insertion sequence (SECIS), located downstream of the Sec codon. Unlike other aminoacyl-tRNAs, Sec-tRNA(Sec) is delivered to the ribosome by a dedicated elongation factor, SelB. We recently identified a series of tRNA(Sec)-like tRNA genes distributed across Bacteria that also encode a canonical tRNA(Sec). These tRNAs contain sequence elements generally recognized by cysteinyl-tRNA synthetase (CysRS). While some of these tRNAs contain a UCA Sec anticodon, most have a GCA Cys anticodon. tRNA(Sec) with GCA anticodons are known to recode UGA codons. Here we investigate the clostridial Desulfotomaculum nigrificans tRNA(Sec)-like tRNA(Cys), and show that this tRNA is acylated by CysRS, recognized by SelB, and capable of UGA recoding with Cys in Escherichia coli. We named this non-canonical group of tRNA(Cys) as tRNA(ReC)' (Recoding with Cys). We performed a comprehensive survey of tRNA(ReC) genes to establish their phylogenetic distribution, and found that, in a particular lineage of clostridial Pelotomaculum, the Cys identity elements of tRNA(ReC) had mutated. This novel tRNA, which contains a UCA anticodon, is capable of Sec incorporation in E. coli, albeit with lower efficiency relative to Pelotomaculum tRNA(Sec). We renamed this unusual tRNA(Sec) derived from tRNA(ReC) as tRNA(ReU)' (Recoding with Sec). Together, our results suggest that tRNA(ReC) and tRNA(ReU) may serve as safeguards in the production of selenoproteins and - to our knowledge - they provide the first example of programmed codon-anticodon mispairing in bacteria.

Background: Development of new aminoacyl-tRNA synthetase (aaRS).tRNA pairs is central for incorporation of novel non-canonical amino acids (ncAAs) into proteins via genetic code expansion (GCE). The Escherichia coli and Caulobacter crescentus histidyl-tRNA synthetases (HisRS) evolved divergent mechanisms of tRNA(His) recognition that prevent their cross-reactivity. Although the E. coil HisRS.tRNA(His) pair is a good candidate for GCE, its use in C. crescentus is limited by the lack of established genetic selection methods and by the low transformation efficiency of C. crescentus. Methods: E. coli was genetically engineered to use a C. crescentus HisRS,tRNA(His) pair. Super-folder green fluorescent protein (sfGFP) and chloramphenicol acetyltransferase (CAT) were used as reporters for read through assays. A library of 313 ncAAs coupled with the sfGFP reporter system was employed to investigate the specificity of E. coil HisRS in vivo. Results: A genomically modified E. coli strain (named MEOV1) was created. MEVO1 requires an active C. crescentus HisRS.wtRNA(His) pair for growth, and displays a similar doubling time as the parental E. coil strain. sfGFP- and CAT-based assays showed that the E. coil HisRS.RNA(His) pair is orthogonal in MEOV1 cells. A mutation in the anticodon loop of E. coli tRNAR(CUA)(His) elevated its suppression efficiency by 2-fold. Conclusions: The C. crescentus HisRS.tRNA(His) pair functionally complements an E. coil Delta hisS strain. The E. coil HisRS.tRNA(His) is orthogonal in MEOV1 cells. E. coil tRNA(CUA)(His) is an efficient amber suppressor in MEOV1. General significance: We developed a platform that allows protein engineering of E. coli HisRS that should facilitate GCE in E. coli. This article is part of a Special Issue entitled "Biochemistry of Synthetic Biology Recent Developments" Guest Editor: Dr. Ilka Heinemann and Dr. Patrick O'Donoghue.

The genetic code—the language used by cells to translate their genomes into proteins that perform many cellular functions—is highly conserved throughout natural life. Rewriting the genetic code could lead to new biological functions such as expanding protein chemistries with noncanonical amino acids (ncAAs) and genetically isolating synthetic organisms from natural organisms and viruses. It has long been possible to transiently produce proteins bearing ncAAs, but stabilizing an expanded genetic code for sustained function in vivo requires an integrated approach: creating recoded genomes and introducing new translation machinery that function together without compromising viability or clashing with endogenous pathways. In this review, we discuss design considerations and technologies for expanding the genetic code. The knowledge obtained by rewriting the genetic code will deepen our understanding of how genomes are designed and how the canonical genetic code evolved.

The human intestine harbors a dense microbial ecosystem (microbiota) that is different between individuals, dynamic over time, and critical for aspects of health and disease. Dietary polysaccharides directly shape the microbiota because of a gap in human digestive physiology, which is equipped to assimilate only proteins, lipids, simple sugars, and starch, leaving nonstarch polysaccharides as major nutrients reaching the microbiota. A mutualistic role of gut microbes is to digest dietary complex carbohydrates, liberating host-absorbable energy via fermentation products. Emerging data indicate that polysaccharides play extensive roles in host–gut microbiota symbiosis beyond dietary polysaccharide digestion, including microbial interactions with endogenous host glycans and the importance of microbial polysaccharides. In this review, we consider multiple mechanisms through which polysaccharides mediate aspects of host-microbe symbiosis in the gut, including some affecting health. As host and microbial metabolic pathways are intimately connected with diet, we highlight the potential to manipulate this system for health.

Cyclic diguanylate (c-di-GMP) is a near universal signaling molecule produced by diguanylate cyclases that can direct a variety of bacterial behaviors. A major area of research over the last several years has been aimed at understanding how a cell with dozens of diguanylate cyclases can deploy a given subset of them to produce a desired phenotypic outcome without undesired cross talk between c-di-GMP-dependent systems. Several models have been put forward to address this question, including specificity of cyclase activation, tuned binding constants of effector proteins, and physical interaction between cyclases and effectors. Additionally, recent evidence has suggested that there may be a link between the catalytic state of a cyclase and its physical contact with an effector. This review highlights several key studies, examines the proposed global and local models of c-di-GMP signaling specificity in bacteria, and attempts to identify the most fruitful steps that can be taken to better understand how dynamic networks of sibling cyclases and effector proteins result in sensible outputs that govern cellular behavior. 179 Click here to view this article's online features: • Download figures as PPT slides • Navigate linked references • Download citations • Explore related articles • Search keywords ANNUAL REVIEWS Further A nn u. R ev . M ic ro bi ol . 2 01 7. 71 :1 79 -1 95 . D ow nl oa de d fr om w w w .a nn ua lr ev ie w s. or g A cc es s pr ov id ed b y D ar tm ou th C ol le ge M ai n L ib ra ry o n 12 /2 7/ 17 . F or p er so na l u se o nl y. MI71CH10-OToole ARI 8 August 2017 9:53

AbstractSelenocystein (Sec oder U) wird durch Neuzuordnung des Stopp‐Codons UGA durch einen Sec‐spezifischen Elongationsfaktor und eine charakteristische RNA‐Struktur codiert. Um mögliche Codonvariationen zu finden, analysierten wir 6.4 Billionen Basenpaare metagenomischer Daten sowie 24 903 mikrobielle Genome für tRNASec‐Spezies. UGA ist erwartungsgemäß das vorherrschende Codon für Sec, allerdings finden wir auch tRNASec‐Spezies, die die Stopp‐Codons UAG und UAA erkennen, sowie weitere zehn Sense‐Codons. Die Synthese von Selenoproteinen durch UAG in Geodermatophilus und Blastococcus sowie durch das Cys‐Codon UGA in Aeromonas salmonicida konnte durch metabolische Markierung mit 75Se oder Massenspektrometrie bestätigt werden. Weitere tRNASec‐Spezies mit verschiedenen Anticodons ermöglichten es Escherichia coli, die aktive Form des Selenoproteins Formiatdehydrogenase H zu synthetisieren. Der genetische Code ist damit bedeutend flexibler, als bisher angenommen.

Selenocysteine (Sec or U) is encoded by UGA, a stop codon reassigned by a Sec-specific elongation factor and a distinctive RNA structure. To discover possible code variations in extant organisms we analyzed 6.4 trillion base pairs of metagenomic sequences and 24 903 microbial genomes for tRNA(Sec) species. As expected, UGA is the predominant Sec codon in use. We also found tRNA(Sec) species that recognize the stop codons UAG and UAA, and ten sense codons. Selenoprotein synthesis programmed by UAG in Geodermatophilus and Blastococcus, and by the Cys codon UGU in Aeromonas salmonicida was confirmed by metabolic labeling with (75) Se or mass spectrometry. Other tRNA(Sec) species with different anticodons enabled E. coli to synthesize active formate dehydrogenase H, a selenoenzyme. This illustrates the ease by which the genetic code may evolve new coding schemes, possibly aiding organisms to adapt to changing environments, and show the genetic code is much more flexible than previously thought.

We report the identification of novel tRNA species with 12-base pair amino-acid acceptor branches composed of longer acceptor stem and shorter T-stem. While canonical tRNAs have a 7/5 configuration of the branch, the novel tRNAs have either 8/4 or 9/3 structure. They were found during the search for selenocysteine tRNAs in terabytes of genome, metagenome and metatranscriptome sequences. Certain bacteria and their phages employ the 8/4 structure for serine and histidine tRNAs, while minor cysteine and selenocysteine tRNA species may have a modified 8/4 structure with one bulge nucleotide. In Acidobacteria, tRNAs with 8/4 and 9/3 structures may function as missense and nonsense suppressor tRNAs and/or regulatory noncoding RNAs. In δ-proteobacteria, an additional cysteine tRNA with an 8/4 structure mimics selenocysteine tRNA and may function as opal suppressor. We examined the potential translation function of suppressor tRNA species in Escherichia coli; tRNAs with 8/4 or 9/3 structures efficiently inserted serine, alanine and cysteine in response to stop and sense codons, depending on the identity element and anticodon sequence of the tRNA. These findings expand our view of how tRNA, and possibly the genetic code, is diversified in nature.

Here we provide the first detailed biochemical study of a noncanonical E1‐like enzyme with broad specificity for cognate ubiquitin‐like (Ubl) proteins that mediates Ubl protein modification and sulfur mobilization to form molybdopterin and thiolated tRNA. Isothermal titration calorimetry and in vivo analyses proved useful in discovering that environmental conditions, ATP binding, and Ubl type controlled the mechanism of association of the Ubl protein with its cognate E1‐like enzyme (SAMP and UbaA of the archaeon Haloferax volcanii, respectively). Further analysis revealed that ATP hydrolysis triggered the formation of thioester and peptide bonds within the Ubl:E1‐like complex. Importantly, the thioester was an apparent precursor to Ubl protein modification but not sulfur mobilization. Comparative modeling to MoeB/ThiF guided the discovery of key residues within the adenylation domain of UbaA that were needed to bind ATP as well as residues that were specifically needed to catalyze the downstream reactions of sulfur mobilization and/or Ubl protein modification. UbaA was also found to be Ubl‐automodified at lysine residues required for early (ATP binding) and late (sulfur mobilization) stages of enzyme activity revealing multiple layers of autoregulation. Cysteine residues, distinct from the canonical E1 ‘active site’ cysteine, were found important in UbaA function supporting a model that this noncanonical E1 is structurally flexible in its active site to allow Ubl~adenylate, Ubl~E1‐like thioester and cysteine persulfide(s) intermediates to form.

AbstractDie Acetylierung von Lysin‐Bausteinen ist eine wichtige posttranslationale Proteinmodifikation. Die Lysin‐Acetylierung in Histonproteinen und deren Interaktion mit anderen posttranslationalen Modifikationen in Histon‐ und nicht‐Histon‐Proteinen ist essentiell bei der DNA‐Replikation, DNA‐Reparatur und Transkriptionsregulation. Wir verwendeten die Flexizym‐Technologie, um die Aminosäuren Acetyllysin (AcK) und nicht‐hydrolysierbares Thioacetyllysin (ThioAcK) in vitro in intakte Proteine einzubauen. ThioAcK und AcK wurden in humanes Histon H3 positionsspezifisch inkorporiert. Dies erfolgte entweder individuell oder paarweise bei verschiedenen Lysin‐Positionen innerhalb des humanen Histon H3. Die Thioacetylgruppe im Histon H3 konnte nicht durch die Histon‐Deacetylase Sirtuin (Typ 1) abgespalten werden, wie durch diese Studie gezeigt wurde. Diese Methode des AcK‐ und ThioAcK‐Einbaus stellt ein wichtiges Werkzeug für die Untersuchung der Protein‐Acetylierung sowie deren Rolle in der Interaktion mit weiteren posttranlationalen Modifizierungen dar.

Acetylation of lysine residues is an important post-translational protein modification. Lysine acetylation in histones and its crosstalk with other post-translational modifications in histone and non-histone proteins are crucial to DNA replication, DNA repair, and transcriptional regulation. We incorporated acetyl-lysine (AcK) and the non-hydrolyzable thioacetyl-lysine (ThioAcK) into full-length proteins in vitro, mediated by flexizyme. ThioAcK and AcK were site-specifically incorporated at different lysine positions into human histone H3, either individually or in pairs. We demonstrate that the thioacetyl group in histone H3 could not be removed by the histone deacetylase sirtuin type 1. This method provides a powerful tool to study protein acetylation and its role in crosstalk between post-translational modifications.

Genetically encoded non-canonical amino acids are powerful tools of protein research and engineering; in particular they allow substitution of individual chemical groups or atoms in a protein of interest. One such amino acid is the tryptophan (Trp) analog 3-benzothienyl-l-alanine (Bta) with an imino-to-sulfur substitution in the five-membered ring. Unlike Trp, Bta is not capable of forming a hydrogen bond, but preserves other properties of a Trp residue. Here we present a pyrrolysyl-tRNA synthetase-derived, engineered enzyme BtaRS that enables efficient and site-specific Bta incorporation into proteins of interest in vivo. Furthermore, we report a 2.1 Å-resolution crystal structure of a BtaRS•Bta complex to show how BtaRS discriminates Bta from canonical amino acids, including Trp. To show utility in protein mutagenesis, we used BtaRS to introduce Bta to replace the Trp28 residue in the active site of Staphylococcus aureus thioredoxin. This experiment showed that not the hydrogen bond between residues Trp28 and Asp58, but the bulky aromatic side chain of Trp28 is important for active site maintenance. Collectively, our study provides a new and robust tool for checking the function of Trp in proteins.

While cytoplasmic tRNA 2-thiolation protein 1 (Tuc1/Ncs6) and ubiquitin-related modifier-1 (Urm1) are important in the 2-thiolation of 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U) at wobble uridines of tRNAs in eukaryotes, the biocatalytic roles and properties of Ncs6/Tuc1 and its homologs are poorly understood. Here we present the first report of an Ncs6 homolog of archaea (NcsA of Haloferax volcanii) that is essential for maintaining cellular pools of thiolated tRNA(Lys)UUU and for growth at high temperature. When purified from Hfx. volcanii, NcsA was found to be modified at Lys204 by isopeptide linkage to polymeric chains of the ubiquitin-fold protein SAMP2. The ubiquitin-activating E1 enzyme homolog of archaea (UbaA) was required for this covalent modification. Non-covalent protein partners that specifically associated with NcsA were also identified including UbaA, SAMP2, proteasome activating nucleotidase (PAN)-A/1, translation elongation factor aEF-1α and a β-CASP ribonuclease homolog of the archaeal cleavage and polyadenylation specificity factor 1 family (aCPSF1). Together, our study reveals that NcsA is essential for growth at high temperature, required for formation of thiolated tRNA(Lys)UUU and intimately linked to homologs of ubiquitin-proteasome, translation and RNA processing systems.

Selenocysteine (Sec) is naturally co-translationally incorporated into proteins by recoding the UGA opal codon with a specialized elongation factor (SelB in bacteria) and an RNA structural signal (SECIS element). We have recently developed a SECIS-free selenoprotein synthesis system that site-specifically--using the UAG amber codon--inserts Sec depending on the elongation factor Tu (EF-Tu). Here, we describe the engineering of EF-Tu for improved selenoprotein synthesis. A Sec-specific selection system was established by expression of human protein O(6)-alkylguanine-DNA alkyltransferase (hAGT), in which the active site cysteine codon has been replaced by the UAG amber codon. The formed hAGT selenoprotein repairs the DNA damage caused by the methylating agent N-methyl-N'-nitro-N-nitrosoguanidine, and thereby enables Escherichia coli to grow in the presence of this mutagen. An EF-Tu library was created in which codons specifying the amino acid binding pocket were randomized. Selection was carried out for enhanced Sec incorporation into hAGT; the resulting EF-Tu variants contained highly conserved amino acid changes within members of the library. The improved UTu-system with EF-Sel1 raises the efficiency of UAG-specific Sec incorporation to >90%, and also doubles the yield of selenoprotein production.

Class I and II aminoacyl‐tRNA synthetases (AARSs) attach amino acids to the 2′‐ and 3′‐OH of the tRNA terminal adenosine, respectively. One exception is phenylalanyl‐tRNA synthetase (PheRS), which belongs to Class II but attaches phenylalanine to the 2′‐OH. Here we show that two Class II AARSs, O‐phosphoseryl‐ (SepRS) and pyrrolysyl‐tRNA (PylRS) synthetases, aminoacylate the 2′‐ and 3′‐OH, respectively. Structure‐based‐phylogenetic analysis reveals that SepRS is more closely related to PheRS than PylRS, suggesting that the idiosyncratic feature of 2′‐OH acylation evolved after the split between PheRS and PylRS. Our work completes the understanding of tRNA aminoacylation positions for the 22 natural AARSs.

The RtcB protein has recently been identified as a 3'-phosphate RNA ligase that directly joins an RNA strand ending with a 2',3'-cyclic phosphate to the 5'-hydroxyl group of another RNA strand in a GTP/Mn(2+)-dependent reaction. Here, we report two crystal structures of Pyrococcus horikoshii RNA-splicing ligase RtcB in complex with Mn(2+) alone (RtcB/ Mn(2+)) and together with a covalently bound GMP (RtcB-GMP/Mn(2+)). The RtcB/ Mn(2+) structure (at 1.6 Å resolution) shows two Mn(2+) ions at the active site, and an array of sulfate ions nearby that indicate the binding sites of the RNA phosphate backbone. The structure of the RtcB-GMP/Mn(2+) complex (at 2.3 Å resolution) reveals the detailed geometry of guanylylation of histidine 404. The critical roles of the key residues involved in the binding of the two Mn(2+) ions, the four sulfates, and GMP are validated in extensive mutagenesis and biochemical experiments, which also provide a thorough characterization for the three steps of the RtcB ligation pathway: (i) guanylylation of the enzyme, (ii) guanylyl-transfer to the RNA substrate, and (iii) overall ligation. These results demonstrate that the enzyme's substrate-induced GTP binding site and the putative reactive RNA ends are in the vicinity of the binuclear Mn(2+) active center, which provides detailed insight into how the enzyme-bound GMP is tansferred to the 3'-phosphate of the RNA substrate for activation and subsequent nucleophilic attack by the 5'-hydroxyl of the second RNA substrate, resulting in the ligated product and release of GMP.

Intron removal from tRNA precursors involves cleavage by a tRNA splicing endonuclease to yield tRNA 3′-halves beginning with a 5′-hydroxyl, and 5′-halves ending in a 2′,3′-cyclic phosphate. A tRNA ligase then incorporates this phosphate into the internucleotide bond that joins the two halves. Although this 3′-P RNA splicing ligase activity was detected almost three decades ago in extracts from animal and later archaeal cells, the protein responsible was not yet identified. Here we report the purification of this ligase from Methanopyrus kandleri cells, and its assignment to the still uncharacterized RtcB protein family. Studies with recombinant Pyrobaculum aerophilum RtcB showed that the enzyme is able to join spliced tRNA halves to mature-sized tRNAs where the joining phosphodiester linkage contains the phosphate originally present in the 2′,3′-cyclic phosphate. The data confirm RtcB as the archaeal RNA 3′-P ligase. Structural genomics efforts previously yielded a crystal structure of the Pyrococcus horikoshii RtcB protein containing a new protein fold and a conserved putative Zn 2+ binding cleft. This structure guided our mutational analysis of the P. aerophilum enzyme. Mutations of highly conserved residues in the cleft (C100A, H205A, H236A) rendered the enzyme inactive suggesting these residues to be part of the active site of the P. aerophilum ligase. There is no significant sequence similarity between the active sites of P. aerophilum ligase and that of T4 RNA ligase, nor ligases from plants and fungi. RtcB sequence conservation in archaea and in eukaryotes implicates eukaryotic RtcB as the long-sought animal 3′-P RNA ligase.

Splicing of mammalian precursor transfer RNA (tRNA) molecules involves two enzymatic steps. First, intron removal by the tRNA splicing endonuclease generates separate 5' and 3' exons. In animals, the second step predominantly entails direct exon ligation by an elusive RNA ligase. Using activity-guided purification of tRNA ligase from HeLa cell extracts, we identified HSPC117, a member of the UPF0027 (RtcB) family, as the essential subunit of a tRNA ligase complex. RNA interference-mediated depletion of HSPC117 inhibited maturation of intron-containing pre-tRNA both in vitro and in living cells. The high sequence conservation of HSPC117/RtcB proteins is suggestive of RNA ligase roles of this protein family in various organisms.

The unfolded protein response (UPR) is an essential signal transduction to cope with protein-folding stress in the endoplasmic reticulum. In the yeast UPR, the unconventional splicing of HAC1 mRNA is a key step. Translation of HAC1 pre-mRNA (HAC1(u) mRNA) is attenuated on polysomes and restarted only after splicing upon the UPR. However, the precise mechanism of this restart remained unclear. Here we show that yeast tRNA ligase (Rlg1p/Trl1p) acting on HAC1 ligation has an unexpected role in HAC1 translation. An RLG1 homologue from Arabidopsis thaliana (AtRLG1) substitutes for yeast RLG1 in tRNA splicing but not in the UPR. Surprisingly, AtRlg1p ligates HAC1 exons, but the spliced mRNA (HAC1(i) mRNA) is not translated efficiently. In the AtRLG1 cells, the HAC1 intron is circularized after splicing and remains associated on polysomes, impairing relief of the translational repression of HAC1(i) mRNA. Furthermore, the HAC1 5' UTR itself enables yeast Rlg1p to regulate translation of the following ORF. RNA IP revealed that yeast Rlg1p is integrated in HAC1 mRNP, before Ire1p cleaves HAC1(u) mRNA. These results indicate that the splicing and the release of translational attenuation of HAC1 mRNA are separable steps and that Rlg1p has pivotal roles in both of these steps.