Co-reporter:Joanne M. Ho, Noah M. Reynolds, Keith Rivera, Morgan Connolly, Li-Tao Guo, Jiqiang Ling, Darryl J. Pappin, George M. Church, and Dieter Söll
ACS Synthetic Biology 2016 Volume 5(Issue 2) pp:163
Publication Date(Web):November 6, 2015
DOI:10.1021/acssynbio.5b00197
Expansion of the genetic code through engineering the translation machinery has greatly increased the chemical repertoire of the proteome. This has been accomplished mainly by read-through of UAG or UGA stop codons by the noncanonical aminoacyl-tRNA of choice. While stop codon read-through involves competition with the translation release factors, sense codon reassignment entails competition with a large pool of endogenous tRNAs. We used an engineered pyrrolysyl-tRNA synthetase to incorporate 3-iodo-l-phenylalanine (3-I-Phe) at a number of different serine and leucine codons in wild-type Escherichia coli. Quantitative LC-MS/MS measurements of amino acid incorporation yields carried out in a selected reaction monitoring experiment revealed that the 3-I-Phe abundance at the Ser208AGU codon in superfolder GFP was 65 ± 17%. This method also allowed quantification of other amino acids (serine, 33 ± 17%; phenylalanine, 1 ± 1%; threonine, 1 ± 1%) that compete with 3-I-Phe at both the aminoacylation and decoding steps of translation for incorporation at the same codon position. Reassignments of different serine (AGU, AGC, UCG) and leucine (CUG) codons with the matching tRNAPyl anticodon variants were met with varying success, and our findings provide a guideline for the choice of sense codons to be reassigned. Our results indicate that the 3-iodo-l-phenylalanyl-tRNA synthetase (IFRS)/tRNAPyl pair can efficiently outcompete the cellular machinery to reassign select sense codons in wild-type E. coli.Keywords: 3-iodo-l-phenylalanyl-tRNA synthetase (IFRS); aminoacyl-tRNA synthetase (aaRS); genetic code; noncanonical amino acid (ncAA); pyrrolysyl-tRNA synthetase (PylRS); quantitative proteomic analysis; selected reaction monitoring (SRM); sense codons reassignment; tRNA competition;
Co-reporter:Rira Kim;Younghoon Lee;Hee-Yoon Lee;Sura Ha;Dieter Söll;Jihye Ahn;Jaehoon Kim;Sungyoon Kim;Aerin Yang;Hee-Sung Park
Science 2016 Volume 354(Issue 6312) pp:623-626
Publication Date(Web):04 Nov 2016
DOI:10.1126/science.aah4428
Radicals push proteins beyond genes
Chemically modifying proteins after their translation can expand their structural and functional roles (see the Perspective by Hofmann and Bode). Two related methods describe how to exploit free radical chemistry to form carbon-carbon bonds between amino acid residues and a selected functional group. Wright et al. added a wide range of functional groups to proteins containing dehydroalanine precursors, with borohydride mediating the radical chemistry. Yang et al. employed a similar approach, using zinc in combination with copper ions. Together, these results will be useful for introducing functionalities and labels to a wide range of proteins.
Science, this issue pp. 597 and 623; see also p. 553
Co-reporter:Dr. Hai Xiong;Dr. Noah M. Reynolds;Dr. Chenguang Fan;Dr. Markus Englert;Dr. Denton Hoyer; Scott J. Miller; Dieter Söll
Angewandte Chemie International Edition 2016 Volume 55( Issue 12) pp:4083-4086
Publication Date(Web):
DOI:10.1002/anie.201511750
Abstract
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.
Co-reporter:Dr. Hai Xiong;Dr. Noah M. Reynolds;Dr. Chenguang Fan;Dr. Markus Englert;Dr. Denton Hoyer; Scott J. Miller; Dieter Söll
Angewandte Chemie 2016 Volume 128( Issue 12) pp:4151-4154
Publication Date(Web):
DOI:10.1002/ange.201511750
Abstract
Die 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.
Co-reporter:Dr. Takahito Mukai;Dr. Markus Englert;Dr. H. James Tripp;Dr. Corwin Miller;Dr. Natalia N. Ivanova;Dr. Edward M. Rubin;Dr. Nikos C. Kyrpides; Dieter Söll
Angewandte Chemie 2016 Volume 128( Issue 17) pp:5423-5427
Publication Date(Web):
DOI:10.1002/ange.201511657
Abstract
Selenocystein (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.
Co-reporter:Dr. Takahito Mukai;Dr. Markus Englert;Dr. H. James Tripp;Dr. Corwin Miller;Dr. Natalia N. Ivanova;Dr. Edward M. Rubin;Dr. Nikos C. Kyrpides; Dieter Söll
Angewandte Chemie International Edition 2016 Volume 55( Issue 17) pp:5337-5341
Publication Date(Web):
DOI:10.1002/anie.201511657
Abstract
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 tRNASec species. As expected, UGA is the predominant Sec codon in use. We also found tRNASec 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 75Se or mass spectrometry. Other tRNASec 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.
Co-reporter:Yuchen Liu;David J. Vinyard;Megan E. Reesbeck;Tateki Suzuki;Kasidet Manakongtreecheep;Patrick L. Holland;Gary W. Brudvig;Dieter Söll
PNAS 2016 113 (45 ) pp:12703-12708
Publication Date(Web):2016-11-08
DOI:10.1073/pnas.1615732113
The sulfur-containing nucleosides in transfer RNA (tRNAs) are present in all three domains of life; they have critical functions
for accurate and efficient translation, such as tRNA structure stabilization and proper codon recognition. The tRNA modification
enzymes ThiI (in bacteria and archaea) and Ncs6 (in archaea and eukaryotic cytosols) catalyze the formation of 4-thiouridine
(s4U) and 2-thiouridine (s2U), respectively. The ThiI homologs were proposed to transfer sulfur via cysteine persulfide enzyme adducts, whereas the reaction
mechanism of Ncs6 remains unknown. Here we show that ThiI from the archaeon Methanococcus maripaludis contains a [3Fe-4S] cluster that is essential for its tRNA thiolation activity. Furthermore, the archaeal and eukaryotic
Ncs6 homologs as well as phosphoseryl-tRNA (Sep-tRNA):Cys-tRNA synthase (SepCysS), which catalyzes the Sep-tRNA to Cys-tRNA
conversion in methanogens, also possess a [3Fe-4S] cluster similar to the methanogenic archaeal ThiI. These results suggest
that the diverse tRNA thiolation processes in archaea and eukaryotic cytosols share a common mechanism dependent on a [3Fe-4S]
cluster for sulfur transfer.
Co-reporter:Isao Tanaka;Min Yao;Kelly Sheppard;Tateki Suzuki;Akiyoshi Nakamura;Koji Kato;Dieter Söll
PNAS 2015 Volume 112 (Issue 2 ) pp:382-387
Publication Date(Web):2015-01-13
DOI:10.1073/pnas.1423314112
Many prokaryotes lack a tRNA synthetase to attach asparagine to its cognate tRNAAsn, and instead synthesize asparagine from tRNAAsn-bound aspartate. This conversion involves two enzymes: a nondiscriminating aspartyl-tRNA synthetase (ND-AspRS) that forms
Asp-tRNAAsn, and a heterotrimeric amidotransferase GatCAB that amidates Asp-tRNAAsn to form Asn-tRNAAsn for use in protein synthesis. ND-AspRS, GatCAB, and tRNAAsn may assemble in an ∼400-kDa complex, known as the Asn-transamidosome, which couples the two steps of asparagine biosynthesis
in space and time to yield Asn-tRNAAsn. We report the 3.7-Å resolution crystal structure of the Pseudomonas aeruginosa Asn-transamidosome, which represents the most common machinery for asparagine biosynthesis in bacteria. We show that, in
contrast to a previously described archaeal-type transamidosome, a bacteria-specific GAD domain of ND-AspRS provokes a principally
new architecture of the complex. Both tRNAAsn molecules in the transamidosome simultaneously serve as substrates and scaffolds for the complex assembly. This architecture
rationalizes an elevated dynamic and a greater turnover of ND-AspRS within bacterial-type transamidosomes, and possibly may
explain a different evolutionary pathway of GatCAB in organisms with bacterial-type vs. archaeal-type Asn-transamidosomes.
Importantly, because the two-step pathway for Asn-tRNAAsn formation evolutionarily preceded the direct attachment of Asn to tRNAAsn, our structure also may reflect the mechanism by which asparagine was initially added to the genetic code.
Co-reporter:Yuchen Liu;Akiyoshi Nakamura;Yuto Nakazawa;Nozomi Asano;Kara A. Ford;Michael J. Hohn;Isao Tanaka;Min Yao;Dieter Söll
PNAS 2014 Volume 111 (Issue 29 ) pp:10520-10525
Publication Date(Web):2014-07-22
DOI:10.1073/pnas.1411267111
Methanogenic archaea lack cysteinyl-tRNA synthetase; they synthesize Cys-tRNA and cysteine in a tRNA-dependent manner. Two
enzymes are required: Phosphoseryl-tRNA synthetase (SepRS) forms phosphoseryl-tRNACys (Sep-tRNACys), which is converted to Cys-tRNACys by Sep-tRNA:Cys-tRNA synthase (SepCysS). This represents the ancestral pathway of Cys biosynthesis and coding in archaea.
Here we report a translation factor, SepCysE, essential for methanococcal Cys biosynthesis; its deletion in Methanococcus maripaludis causes Cys auxotrophy. SepCysE acts as a scaffold for SepRS and SepCysS to form a stable high-affinity complex for tRNACys causing a 14-fold increase in the initial rate of Cys-tRNACys formation. Based on our crystal structure (2.8-Å resolution) of a SepCysS⋅SepCysE complex, a SepRS⋅SepCysE⋅SepCysS structure
model suggests that this ternary complex enables substrate channeling of Sep-tRNACys. A phylogenetic analysis suggests coevolution of SepCysE with SepRS and SepCysS in the last universal common ancestral state.
Our findings suggest that the tRNA-dependent Cys biosynthesis proceeds in a multienzyme complex without release of the intermediate
and this mechanism may have facilitated the addition of Cys to the genetic code.
Co-reporter:Margaret Wong;Li-Tao Guo;Patrick O’Donoghue;Akiyoshi Nakamura;Yane-Shih Wang;Jennifer M. Kavran;Daniel Eiler;Thomas A. Steitz;Laura L. Kiessling;Dieter Söll
PNAS 2014 Volume 111 (Issue 47 ) pp:16724-16729
Publication Date(Web):2014-11-25
DOI:10.1073/pnas.1419737111
Pyrrolysyl-tRNA synthetase (PylRS) and its cognate tRNAPyl have emerged as ideal translation components for genetic code innovation. Variants of the enzyme facilitate the incorporation
>100 noncanonical amino acids (ncAAs) into proteins. PylRS variants were previously selected to acylate Nε-acetyl-Lys (AcK) onto tRNAPyl. Here, we examine an Nε-acetyl-lysyl-tRNA synthetase (AcKRS), which is polyspecific (i.e., active with a broad range of ncAAs) and 30-fold more efficient
with Phe derivatives than it is with AcK. Structural and biochemical data reveal the molecular basis of polyspecificity in
AcKRS and in a PylRS variant [iodo-phenylalanyl-tRNA synthetase (IFRS)] that displays both enhanced activity and substrate
promiscuity over a chemical library of 313 ncAAs. IFRS, a product of directed evolution, has distinct binding modes for different
ncAAs. These data indicate that in vivo selections do not produce optimally specific tRNA synthetases and suggest that translation
fidelity will become an increasingly dominant factor in expanding the genetic code far beyond 20 amino acids.
Co-reporter:Dr. Markus J. Bröcker;Joanne M. L. Ho; George M. Church; Dieter Söll; Patrick O'Donoghue
Angewandte Chemie 2014 Volume 126( Issue 1) pp:325-330
Publication Date(Web):
DOI:10.1002/ange.201308584
Abstract
Der Selenocystein(Sec)-Einbau in Proteine erfolgt in der Natur durch die kotranslationale Umkodierung eines UGA-Stopp-Codons. Diese Studie zeigt nun, dass Sec nicht ausschließlich durch UGA, sondern vielmehr durch 58 aller 64 möglichen Codons kodiert werden kann. Hierbei erlauben 15 Codons nicht nur die vollständige Umkodierung von ihrer ursprünglichen Bedeutung als kanonische Aminosäure zu Selenocystein, sondern sie führen zu Proteinausbeuten, die um mehr als das Zehnfache gesteigert sind. Der hoch effiziente Mechanismus zur Selenocystein-Rekodierung wird anhand zweier Reporterenzyme, der Escherichia-coli-Formiatdehydrogenase und der humanen Thioredoxinreduktase, beschrieben. Da der Selenocysteineinbau an der Position eines UGA-Stopp-Codons zwangsläufig mit translationaler Termination konkurriert, war es umso erstaunlicher, dass die Selenocystein-Einbaumaschinerie während der Umkodierung von Sense-Codons erfolgreich mit den im Überfluss vorhandenen regulären Aminoacyl-tRNAs konkurriert.
Co-reporter:Dr. Markus J. Bröcker;Joanne M. L. Ho; George M. Church; Dieter Söll; Patrick O'Donoghue
Angewandte Chemie 2014 Volume 126( Issue 1) pp:
Publication Date(Web):
DOI:10.1002/ange.201310509
Co-reporter:Dr. Markus J. Bröcker;Joanne M. L. Ho; George M. Church; Dieter Söll; Patrick O'Donoghue
Angewandte Chemie International Edition 2014 Volume 53( Issue 1) pp:319-323
Publication Date(Web):
DOI:10.1002/anie.201308584
Abstract
Selenocysteine (Sec) is naturally incorporated into proteins by recoding the stop codon UGA. Sec is not hardwired to UGA, as the Sec insertion machinery was found to be able to site-specifically incorporate Sec directed by 58 of the 64 codons. For 15 sense codons, complete conversion of the codon meaning from canonical amino acid (AA) to Sec was observed along with a tenfold increase in selenoprotein yield compared to Sec insertion at the three stop codons. This high-fidelity sense-codon recoding mechanism was demonstrated for Escherichia coli formate dehydrogenase and recombinant human thioredoxin reductase and confirmed by independent biochemical and biophysical methods. Although Sec insertion at UGA is known to compete against protein termination, it is surprising that the Sec machinery has the ability to outcompete abundant aminoacyl-tRNAs in decoding sense codons. The findings have implications for the process of translation and the information storage capacity of the biological cell.
Co-reporter:Dr. Markus J. Bröcker;Joanne M. L. Ho; George M. Church; Dieter Söll; Patrick O'Donoghue
Angewandte Chemie International Edition 2014 Volume 53( Issue 1) pp:
Publication Date(Web):
DOI:10.1002/anie.201310509
Co-reporter:Martin Kucklick;Patrick O’Donoghue;Dieter Söll;Johannes G. Schäfer;Katharina Riedel;Ilka U. Heinemann;Jesse Rinehart;Laure Prat
PNAS 2014 Volume 111 (Issue 48 ) pp:17206-17211
Publication Date(Web):2014-12-02
DOI:10.1073/pnas.1420193111
Expanding the genetic code is an important aim of synthetic biology, but some organisms developed naturally expanded genetic
codes long ago over the course of evolution. Less than 1% of all sequenced genomes encode an operon that reassigns the stop
codon UAG to pyrrolysine (Pyl), a genetic code variant that results from the biosynthesis of Pyl-tRNAPyl. To understand the selective advantage of genetically encoding more than 20 amino acids, we constructed a markerless tRNAPyl deletion strain of Methanosarcina acetivorans (ΔpylT) that cannot decode UAG as Pyl or grow on trimethylamine. Phenotypic defects in the ΔpylT strain were evident in minimal medium containing methanol. Proteomic analyses of wild type (WT) M. acetivorans and ΔpylT cells identified 841 proteins from >7,000 significant peptides detected by MS/MS. Protein production from UAG-containing
mRNAs was verified for 19 proteins. Translation of UAG codons was verified by MS/MS for eight proteins, including identification
of a Pyl residue in PylB, which catalyzes the first step of Pyl biosynthesis. Deletion of tRNAPyl globally altered the proteome, leading to >300 differentially abundant proteins. Reduction of the genetic code from 21 to
20 amino acids led to significant down-regulation in translation initiation factors, amino acid metabolism, and methanogenesis
from methanol, which was offset by a compensatory (100-fold) up-regulation in dimethyl sulfide metabolic enzymes. The data
show how a natural proteome adapts to genetic code reduction and indicate that the selective value of an expanded genetic
code is related to carbon source range and metabolic efficiency.
Co-reporter:Dr. Chenguang Fan;Joanne M. L. Ho;Napon Chirathivat; Dr. Dieter Söll;Dr. Yane-Shih Wang
ChemBioChem 2014 Volume 15( Issue 12) pp:1805-1809
Publication Date(Web):
DOI:10.1002/cbic.201402083
Abstract
We tested the substrate range of four wild-type E. coli aminoacyl-tRNA synthetases (AARSs) with a library of nonstandard amino acids (nsAAs). Although these AARSs could discriminate efficiently against the other canonical amino acids, they were able to use many nsAAs as substrates. Our results also show that E. coli tryptophanyl-tRNA synthetase (TrpRS) and tyrosyl-tRNA synthetase have overlapping substrate ranges. In addition, we found that the nature of the anticodon sequence of tRNATrp altered the nsAA substrate range of TrpRS; this implies that the sequence of the anticodon affects the TrpRS amino acid binding pocket. These results highlight again that inherent AARS polyspecificity will be a major challenge in the aim of incorporating multiple different amino acids site-specifically into proteins.
Co-reporter:Dr. Radha Krishnakumar;Dr. Laure Prat;Dr. Hans-Rudolf Aerni;Dr. Jiqiang Ling; Dr. Chuck Merryman; John I. Glass; Jesse Rinehart; Dieter Söll
ChemBioChem 2013 Volume 14( Issue 15) pp:1967-1972
Publication Date(Web):
DOI:10.1002/cbic.201300444
Abstract
Sense codon recoding is the basis for genetic code expansion with more than two different noncanonical amino acids. It requires an unused (or rarely used) codon, and an orthogonal tRNA synthetase:tRNA pair with the complementary anticodon. The Mycoplasma capricolum genome contains just six CGG arginine codons, without a dedicated tRNAArg. We wanted to reassign this codon to pyrrolysine by providing M. capricolum with pyrrolysyl-tRNA synthetase, a synthetic tRNA with a CCG anticodon (), and the genes for pyrrolysine biosynthesis. Here we show that is efficiently recognized by the endogenous arginyl-tRNA synthetase, presumably at the anticodon. Mass spectrometry revealed that in the presence of , CGG codons are translated as arginine. This result is not unexpected as most tRNA synthetases use the anticodon as a recognition element. The data suggest that tRNA misidentification by endogenous aminoacyl-tRNA synthetases needs to be overcome for sense codon recoding.
Co-reporter:Markus J. Bröcker;Yuzuru Itoh;Shun-ichi Sekine;Gifty Hammond;Shiro Suetsugu;Dieter Söll;Shigeyuki Yokoyama
Science 2013 Volume 340(Issue 6128) pp:75-78
Publication Date(Web):05 Apr 2013
DOI:10.1126/science.1229521
Putting Selenium in Proteins
The 21st amino acid, selenocysteine (Sec), occurs in the active site of many redox enzymes. Its cognate transfer RNA (tRNA) is first loaded with Ser by seryl-tRNA synthetase and the Ser-tRNASec is then converted to Sec-tRNASec. Itoh et al. (p. 75) determined the crystal structures of the selenocysteine synthase, SelA, that is responsible for this conversion in bacteria, alone and in complex with tRNA. The decameric SelA complex binds to 10 tRNASec molecules. The structures, together with biochemistry, show how SelA discriminates tRNASec from tRNASer, give insight into the mechanism of catalysis, and show that decamerization is essential for function.
Co-reporter:Alexander Sczyrba;James H. Campbell;Alisha G. Campbell;Patrick Schwientek;Tanja Woyke;Patrick O’Donoghue;Mircea Podar;Dieter Söll
PNAS 2013 Volume 110 (Issue 14 ) pp:5540-5545
Publication Date(Web):2013-04-02
DOI:10.1073/pnas.1303090110
The composition of the human microbiota is recognized as an important factor in human health and disease. Many of our cohabitating
microbes belong to phylum-level divisions for which there are no cultivated representatives and are only represented by small
subunit rRNA sequences. For one such taxon (SR1), which includes bacteria with elevated abundance in periodontitis, we provide
a single-cell genome sequence from a healthy oral sample. SR1 bacteria use a unique genetic code. In-frame TGA (opal) codons
are found in most genes (85%), often at loci normally encoding conserved glycine residues. UGA appears not to function as
a stop codon and is in equilibrium with the canonical GGN glycine codons, displaying strain-specific variation across the
human population. SR1 encodes a divergent tRNAGlyUCA with an opal-decoding anticodon. SR1 glycyl-tRNA synthetase acylates tRNAGlyUCA with glycine in vitro with similar activity compared with normal tRNAGlyUCC. Coexpression of SR1 glycyl-tRNA synthetase and tRNAGlyUCA in Escherichia coli yields significant β-galactosidase activity in vivo from a lacZ gene containing an in-frame TGA codon. Comparative genomic analysis with Human Microbiome Project data revealed that the
human body harbors a striking diversity of SR1 bacteria. This is a surprising finding because SR1 is most closely related
to bacteria that live in anoxic and thermal environments. Some of these bacteria share common genetic and metabolic features
with SR1, including UGA to glycine reassignment and an archaeal-type ribulose-1,5-bisphosphate carboxylase (RubisCO) involved
in AMP recycling. UGA codon reassignment renders SR1 genes untranslatable by other bacteria, which impacts horizontal gene
transfer within the human microbiota.
Co-reporter:Caroline Aldag;Markus J. Bröcker;Michael J. Hohn;Laure Prat;Gifty Hammond;Abigail Plummer ;Dieter Söll
Angewandte Chemie International Edition 2013 Volume 52( Issue 5) pp:1441-1445
Publication Date(Web):
DOI:10.1002/anie.201207567
Co-reporter:Caroline Aldag;Markus J. Bröcker;Michael J. Hohn;Laure Prat;Gifty Hammond;Abigail Plummer ;Dieter Söll
Angewandte Chemie 2013 Volume 125( Issue 5) pp:1481-1485
Publication Date(Web):
DOI:10.1002/ange.201207567
Co-reporter:Taiki Nemoto;Akiyoshi Nakamura;Keisuke Komoda;Ilka U. Heinemann;Min Yao;Keitaro Yamashita;Tomoyo Sonoda;Dieter Söll;Isao Tanaka
PNAS 2013 Volume 110 (Issue 52 ) pp:20970-20975
Publication Date(Web):2013-12-24
DOI:10.1073/pnas.1321312111
Nucleotide polymerization proceeds in the forward (5′-3′) direction. This tenet of the central dogma of molecular biology
is found in diverse processes including transcription, reverse transcription, DNA replication, and even in lagging strand
synthesis where reverse polymerization (3′-5′) would present a “simpler” solution. Interestingly, reverse (3′-5′) nucleotide
addition is catalyzed by the tRNA maturation enzyme tRNAHis guanylyltransferase, a structural homolog of canonical forward polymerases. We present a Candida albicans tRNAHis guanylyltransferase-tRNAHis complex structure that reveals the structural basis of reverse polymerization. The directionality of nucleotide polymerization
is determined by the orientation of approach of the nucleotide substrate. The tRNA substrate enters the enzyme’s active site
from the opposite direction (180° flip) compared with similar nucleotide substrates of canonical 5′-3′ polymerases, and the
finger domains are on opposing sides of the core palm domain. Structural, biochemical, and phylogenetic data indicate that
reverse polymerization appeared early in evolution and resembles a mirror image of the forward process.
Co-reporter:Caroline Aldag;Markus J. Bröcker;Michael J. Hohn;Laure Prat;Gifty Hammond;Abigail Plummer ;Dieter Söll
Angewandte Chemie 2013 Volume 125( Issue 5) pp:
Publication Date(Web):
DOI:10.1002/ange.201300063
Co-reporter:Caroline Aldag;Markus J. Bröcker;Michael J. Hohn;Laure Prat;Gifty Hammond;Abigail Plummer ;Dieter Söll
Angewandte Chemie International Edition 2013 Volume 52( Issue 5) pp:
Publication Date(Web):
DOI:10.1002/anie.201300063
Co-reporter:Jiqiang Ling;Ivana Simonović;Chris Cho;Kaitlyn M. Peterson;Miljan Simonović;Dieter Söll
PNAS 2012 Volume 109 (Issue 9 ) pp:
Publication Date(Web):2012-02-28
DOI:10.1073/pnas.1200109109
Aminoacyl-tRNA synthetases (aaRSs) ensure faithful translation of mRNA into protein by coupling an amino acid to a set of
tRNAs with conserved anticodon sequences. Here, we show that in mitochondria of Saccharomyces cerevisiae, a single aaRS (MST1) recognizes and aminoacylates two natural tRNAs that contain anticodon loops of different size and sequence.
Besides a regular with a threonine (Thr) anticodon, MST1 also recognizes an unusual , which contains an enlarged anticodon loop and an anticodon triplet that reassigns the CUN codons from leucine to threonine.
Our data show that MST1 recognizes the anticodon loop in both tRNAs, but employs distinct recognition mechanisms. The size
but not the sequence of the anticodon loop is critical for recognition, whereas the anticodon sequence is essential for aminoacylation of . The crystal structure of MST1 reveals that, while lacking the N-terminal editing domain, the enzyme closely resembles the
bacterial threonyl-tRNA synthetase (ThrRS). A detailed structural comparison with Escherichia coli ThrRS, which is unable to aminoacylate , reveals differences in the anticodon-binding domain that probably allow recognition of the distinct anticodon loops. Finally,
our mutational and modeling analyses identify the structural elements in MST1 (e.g., helix α11) that define tRNA selectivity.
Thus, MTS1 exemplifies that a single aaRS can recognize completely divergent anticodon loops of natural isoacceptor tRNAs
and that in doing so it facilitates the reassignment of the genetic code in yeast mitochondria.
Co-reporter:Jesse Rinehart;Laure Prat;Ilka U. Heinemann;Patrick O’Donoghue;Dieter Söll;Hans R. Aerni
PNAS 2012 Volume 109 (Issue 51 ) pp:21070-21075
Publication Date(Web):2012-12-18
DOI:10.1073/pnas.1218613110
Despite the fact that the genetic code is known to vary between organisms in rare cases, it is believed that in the lifetime
of a single cell the code is stable. We found Acetohalobium arabaticum cells grown on pyruvate genetically encode 20 amino acids, but in the presence of trimethylamine (TMA), A. arabaticum dynamically expands its genetic code to 21 amino acids including pyrrolysine (Pyl). A. arabaticum is the only known organism that modulates the size of its genetic code in response to its environment and energy source.
The gene cassette pylTSBCD, required to biosynthesize and genetically encode UAG codons as Pyl, is present in the genomes of 24 anaerobic archaea and
bacteria. Unlike archaeal Pyl-decoding organisms that constitutively encode Pyl, we observed that A. arabaticum controls Pyl encoding by down-regulating transcription of the entire Pyl operon under growth conditions lacking TMA, to the
point where no detectable Pyl-tRNAPyl is made in vivo. Pyl-decoding archaea adapted to an expanded genetic code by minimizing TAG codon frequency to typically
∼5% of ORFs, whereas Pyl-decoding bacteria (∼20% of ORFs contain in-frame TAGs) regulate Pyl-tRNAPyl formation and translation of UAG by transcriptional deactivation of genes in the Pyl operon. We further demonstrate that
Pyl encoding occurs in a bacterium that naturally encodes the Pyl operon, and identified Pyl residues by mass spectrometry
in A. arabaticum proteins including two methylamine methyltransferases.
Co-reporter:Chiaki Okada;Markus Englert;Shuangluo Xia;Soo Hyun Eom;Min Yao;Ved Tanavde;Jimin Wang;Akiyoshi Nakamura;William H. Konigsberg;Dieter Söll
PNAS 2012 Volume 109 (Issue 38 ) pp:15235-15240
Publication Date(Web):2012-09-18
DOI:10.1073/pnas.1213795109
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/Mn2+-dependent reaction. Here, we report two crystal structures of Pyrococcus horikoshii RNA-splicing ligase RtcB in complex with Mn2+ alone (RtcB/ Mn2+) and together with a covalently bound GMP (RtcB-GMP/Mn2+). The RtcB/ Mn2+ structure (at 1.6 Å resolution) shows two Mn2+ 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/Mn2+ 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 Mn2+ 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 Mn2+ 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.
Co-reporter:Hee-Sung Park;Michael J. Hohn;Takuya Umehara;Li-Tao Guo;Edith M. Osborne;Jack Benner;Christopher J. Noren;Jesse Rinehart;Dieter Söll
Science 2011 Volume 333(Issue 6046) pp:1151-1154
Publication Date(Web):26 Aug 2011
DOI:10.1126/science.1207203
Engineered bacterial translation can be used to direct site-specific insertion of an amino acid into proteins.
Co-reporter:Patrick O’Donoghue;Kelly Sheppard;Osamu Nureki;Dieter Söll
PNAS 2011 108 (51 ) pp:
Publication Date(Web):2011-12-20
DOI:10.1073/pnas.1117294108
The specificity of most aminoacyl-tRNA synthetases for an amino acid and cognate tRNA pair evolved before the divergence of
the three domains of life. Glutaminyl-tRNA synthetase (GlnRS) evolved later and is derived from the archaeal-type nondiscriminating
glutamyl-tRNA synthetase (GluRS), an enzyme with relaxed tRNA specificity capable of forming both Glu-tRNAGlu and Glu-tRNAGln. The archaea lack GlnRS and use a specialized amidotransferase to convert Glu-tRNAGln to Gln-tRNAGln needed for protein synthesis. We show that the Methanothermobacter thermautotrophicus GluRS is active toward tRNAGlu and the two tRNAGln isoacceptors the organism encodes, but with a significant catalytic preference for . The less active responds to the less common CAA codon for Gln. From a biochemical characterization of M. thermautotrophicus GluRS variants, we found that the evolution of tRNA specificity in GlnRS could be recapitulated by converting the M. thermautotrophicus GluRS to a tRNAGln specific enzyme, solely through the addition of an acceptor stem loop present in bacterial GlnRS. One designed GluRS variant
is also highly specific for the isoacceptor, which responds to the CAG codon, and shows no activity toward . Because it is now possible to eliminate particular codons from the genome of Escherichia coli, additional codons will become available for genetic code engineering. Isoacceptor-specific aminoacyl-tRNA synthetases will
enable the reassignment of more open codons while preserving accurate encoding of the 20 canonical amino acids.
Co-reporter:Dieter Söll;Cindy L. Will;Simon Trowitzsch;Alexander Schleiffer;Johannes Popow;Stefan Weitzer;Javier Martinez;Karl Mechtler;Reinhard Lührmann;Markus Englert;Beata Mierzwa
Science 2011 Volume 331(Issue 6018) pp:760-764
Publication Date(Web):11 Feb 2011
DOI:10.1126/science.1197847
The human enzyme that joins transfer RNA exons together is discovered.
Co-reporter:Dieter Söll;Jiqiang Ling
PNAS 2010 Volume 107 (Issue 9 ) pp:4028-4033
Publication Date(Web):2010-03-02
DOI:10.1073/pnas.1000315107
Oxidative stress arises from excessive reactive oxygen species (ROS) and affects organisms of all three domains of life. Here
we present a previously unknown pathway through which ROS may impact faithful protein synthesis. Aminoacyl-tRNA synthetases
are key enzymes in the translation of the genetic code; they attach the correct amino acid to each tRNA species and hydrolyze
an incorrectly attached amino acid in a process called editing. We show both in vitro and in vivo in Escherichia coli that ROS reduced the overall translational fidelity by impairing the editing activity of threonyl-tRNA synthetase. Hydrogen
peroxide oxidized cysteine182 residue critical for editing, leading to Ser-tRNAThr formation and protein mistranslation that impaired growth of Escherichia coli. The presence of major heat shock proteases was required to allow cell growth in medium containing serine and hydrogen peroxide;
this suggests that the mistranslated proteins were misfolded.
Co-reporter:Sarath Gundllapalli;Markus Englert;Kelly Sheppard;Hildburg Beier;Dieter Söll
PNAS 2010 Volume 107 (Issue 39 ) pp:16834-16839
Publication Date(Web):2010-09-28
DOI:10.1073/pnas.1011703107
Animal cells have two tRNA splicing pathways: (i) a 5′-P ligation mechanism, where the 5′-phosphate of the 3′ tRNA half becomes the junction phosphate of the new phosphodiester
linkage, and (ii) a 3′-P ligation process, in which the 3′-phosphate of the 5′ tRNA half turns into the junction phosphate. Although both
activities are known to exist in animals, in almost three decades of investigation, neither of the two RNA ligases has been
identified. Here we describe a gene from the chordate Branchiostoma floridae that encodes an RNA ligase (Bf RNL) with a strict requirement for RNA substrates with a 2′-phosphate terminus for the ligation
of RNAs with 5′-phosphate and 3′-hydroxyl ends. Unlike the yeast and plant tRNA ligases involved in tRNA splicing, Bf RNL
lacks healing activities and requires the action of a polynucleotide kinase (PNK) and a cyclic phosphodiesterase (CDPase)
in trans. The activities of these two enzymes were identified in a single B. floridae protein (Bf PNK/CPDase). The combined activities of Bf RNL and Bf PNK/CPDase are sufficient for the joining of tRNA splicing
intermediates in vitro, and for the functional complementation of a tRNA ligase-deficient Saccharomyces cerevisiae strain in vivo. Hence, these two proteins constitute the 5′-P RNA ligation pathway in an animal organism.
Co-reporter:Kayo Nozawa,
Patrick O'Donoghue,
Sarath Gundllapalli,
Yuhei Araiso,
Ryuichiro Ishitani,
Takuya Umehara,
Dieter Söll
&
Osamu Nureki
Nature 2009 457(7233) pp:1163
Publication Date(Web):2008-12-31
DOI:10.1038/nature07611
Pyrrolysine (Pyl), the 22nd natural amino acid, is genetically encoded by UAG and inserted into proteins by the unique suppressor tRNAPyl (ref. 1). The Methanosarcinaceae produce Pyl and express Pyl-containing methyltransferases that allow growth on methylamines2. Homologous methyltransferases and the Pyl biosynthetic and coding machinery are also found in two bacterial species1, 3. Pyl coding is maintained by pyrrolysyl-tRNA synthetase (PylRS), which catalyses the formation of Pyl-tRNAPyl (refs 4, 5). Pyl is not a recent addition to the genetic code. PylRS was already present in the last universal common ancestor6; it then persisted in organisms that utilize methylamines as energy sources. Recent protein engineering efforts added non-canonical amino acids to the genetic code7, 8. This technology relies on the directed evolution of an ‘orthogonal’ tRNA synthetase–tRNA pair in which an engineered aminoacyl-tRNA synthetase (aaRS) specifically and exclusively acylates the orthogonal tRNA with a non-canonical amino acid. For Pyl the natural evolutionary process developed such a system some 3 billion years ago. When transformed into Escherichia coli, Methanosarcina barkeri PylRS and tRNAPyl function as an orthogonal pair in vivo5, 9. Here we show that Desulfitobacterium hafniense PylRS–tRNAPyl is an orthogonal pair in vitro and in vivo, and present the crystal structure of this orthogonal pair. The ancient emergence of PylRS–tRNAPyl allowed the evolution of unique structural features in both the protein and the tRNA. These structural elements manifest an intricate, specialized aaRS–tRNA interaction surface that is highly distinct from those observed in any other known aaRS–tRNA complex; it is this general property that underlies the molecular basis of orthogonality.
Co-reporter:Sotiria Palioura;Janine Marazzi;Eric Aeby;Mascha Pusnik;Allyson Lieberman;André Schneider;Elisabetta Ullu;Dieter Söll
PNAS 2009 Volume 106 (Issue 13 ) pp:5088-5092
Publication Date(Web):2009-03-31
DOI:10.1073/pnas.0901575106
The micronutrient selenium is found in proteins as selenocysteine (Sec), the 21st amino acid cotranslationally inserted in
response to a UGA codon. In vitro studies in archaea and mouse showed that Sec-tRNASec formation is a 3-step process starting with serylation of tRNASec by seryl-tRNA synthetase (SerRS), phosphorylation of serine to form phosphoserine (Sep)-tRNASec by phosphoseryl-tRNASec kinase (PSTK), and conversion to Sec-tRNASec by Sep-tRNA:Sec-tRNA synthase (SepSecS). However, a complete study of eukaryotic selenoprotein synthesis has been lacking.
Here, we present an analysis of Sec-tRNASec formation in the parasitic protozoon Trypanosoma brucei in vivo. Null mutants of either PSTK or SepSecS abolished selenoprotein synthesis, demonstrating the essentiality of both
enzymes for Sec-tRNASec formation. Growth of the 2 knockout strains was not impaired; thus, unlike mammals, trypanosomes do not require selenoproteins
for viability. Analysis of conditional RNAi strains showed that SerRS, selenophosphate synthase, and the Sec-specific elongation
factor, EFSec, are also essential for selenoprotein synthesis. These results with T. brucei imply that eukaryotes have a single pathway of Sec-tRNASec synthesis that requires Sep-tRNASec as an intermediate.
Co-reporter:Catherine Madinger;Ilka U. Heinemann;Jack Benner;Patrick O'Donoghue;Dieter Söll;Lennart Randau;Christopher J. Noren
PNAS 2009 Volume 106 (Issue 50 ) pp:21103-21108
Publication Date(Web):2009-12-15
DOI:10.1073/pnas.0912072106
tRNAHis guanylyltransferase (Thg1) post-transcriptionally adds a G (position −1) to the 5′-terminus of tRNAHis. The Methanosarcina acetivorans Thg1 (MaThg1) gene contains an in-frame TAG (amber) codon. Although a UAG codon typically directs translation termination,
its presence in Methanosarcina mRNA may lead to pyrrolysine (Pyl) incorporation achieved by Pyl-tRNAPyl, the product of pyrrolysyl-tRNA synthetase. Sequencing of the MaThg1 gene and transcript confirmed the amber codon. Translation
of MaThg1 mRNA led to a full-length, Pyl-containing, active enzyme as determined by immunoblotting, mass spectrometry, and
biochemical analysis. The nature of the inserted amino acid at the position specified by UAG is not critical, as Pyl or Trp
insertion yields active MaThg1 variants in M. acetivorans and equal amounts of full-length protein. These data suggest that Pyl insertion is akin to natural suppression and unlike
the active stop codon reassignment that is required for selenocysteine insertion. Only three Pyl-containing proteins have
been characterized previously, a set of methylamine methyltransferases in which Pyl is assumed to have specifically evolved
to be a key active-site constituent. In contrast, Pyl in MaThg1 is a dispensable residue that appears to confer no selective
advantage. Phylogenetic analysis suggests that Thg1 is becoming dispensable in the archaea, and furthermore supports the hypothesis
that Pyl appeared in MaThg1 as the result of neutral evolution. This indicates that even the most unusual amino acid can play
an ordinary role in proteins.
Co-reporter:Yuhei Araiso;R. Lynn Sherrer;Ryuichiro Ishitani;Joanne M. L. Ho;Dieter Söll;Osamu Nureki
PNAS 2009 Volume 106 (Issue 38 ) pp:16215-16220
Publication Date(Web):2009-09-22
DOI:10.1073/pnas.0908861106
Compared to bacteria, archaea and eukaryotes employ an additional enzyme for the biosynthesis of selenocysteine (Sec), the
21st natural amino acid (aa). An essential RNA-dependent kinase, O-phosphoseryl-tRNASec kinase (PSTK), converts seryl-tRNASec to O-phosphoseryl-tRNASec, the immediate precursor of selenocysteinyl-tRNASec. The sequence of Methanocaldococcus jannaschii PSTK (MjPSTK) suggests an N-terminal kinase domain (177 aa) followed by a presumed tRNA binding region (75 aa). The structures
of MjPSTK complexed with ADP and AMPPNP revealed that this enzyme belongs to the P-loop kinase class, and that the kinase
domain is closely related to gluconate kinase and adenylate kinase. ATP is bound by the P-loop domain (residues 11–18). Formed
by antiparallel dimerization of two PSTK monomers, the enzyme structure shows a deep groove with positive electrostatic potential.
Located in this groove is the enzyme's active site, which biochemical and genetic data suggest is composed of Asp-41, Arg-44,
Glu-55, Tyr-82, Tyr-83, Met-86, and Met-132. Based on structural comparison with Escherichia coli adenylate kinase a docking model was generated that assigns these amino acids to the recognition of the terminal A76-Ser
moieties of Ser-tRNASec. The geometry and electrostatic environment of the groove in MjPSTK are perfectly complementary to the unusually long acceptor
helix of tRNASec.
Co-reporter:Andrew Kohlway;Bradford J. Stanley;Yong Xiong;Dieter Söll;Sarah Mechta;Lennart Randau
Science 2009 Volume 324(Issue 5927) pp:657-659
Publication Date(Web):01 May 2009
DOI:10.1126/science.1170123
Co-reporter:Sotiria Palioura;R. Lynn Sherrer;Thomas A. Steitz;Dieter Söll;Miljan Simonović
Science 2009 Volume 325(Issue 5938) pp:321-325
Publication Date(Web):17 Jul 2009
DOI:10.1126/science.1173755
Co-reporter:Jing Yuan;Kelly Sheppard;Dieter Söll
Acta Biochimica et Biophysica Sinica 2008 Volume 40( Issue 7) pp:539-553
Publication Date(Web):
DOI:10.1111/j.1745-7270.2008.00435.x
The accurate formation of cognate aminoacyl-transfer RNAs (aa-tRNAs) is essential for the fidelity of translation. Most amino acids are esterified onto their cognate tRNA isoacceptors directly by aa-tRNA synthetases. However, in the case of four amino acids (Gln, Asn, Cys and Sec), aminoacyl-tRNAs are made through indirect pathways in many organisms across all three domains of life. The process begins with the charging of noncognate amino acids to tRNAs by a specialized synthetase in the case of Cys-tRNACys formation or by synthetases with relaxed specificity, such as the non-di scr iminating glutamyl-tRNA, non-disc rimi nati ng aspartyl-tRNA and seryl-tRNA synthetases. The resulting misacylated tRNAs are then converted to cognate pairs through transformation of the amino acids on the tRNA, which is catalyzed by a group of tRNA-dependent modifying enzymes, such as tRNA-dependent amidotransferases, Sep-tRNA:Cys-tRNA synthase, O-phosphoseryl-tRNA kinase and Sep-tRNA:Sec-tRNA synthase. The majority of these indirect pathways are widely spread in all domains of life and thought to be part of the evolutionary process.
Co-reporter:Lennart Randau,
Imke Schröder
&
Dieter Söll
Nature 2008 453(7191) pp:120
Publication Date(Web):2008-05-01
DOI:10.1038/nature06833
The universality of ribonuclease P (RNase P), the ribonucleoprotein essential for transfer RNA (tRNA) 5′ maturation1, 2, is challenged in the archaeon Nanoarchaeum equitans. Neither extensive computational analysis of the genome nor biochemical tests in cell extracts revealed the existence of this enzyme. Here we show that the conserved placement of its tRNA gene promoters allows the synthesis of leaderless tRNAs, whose presence was verified by the observation of 5′ triphosphorylated mature tRNA species. Initiation of tRNA gene transcription requires a purine, which coincides with the finding that tRNAs with a cytosine in position 1 display unusually extended 5′ termini with an extra purine residue. These tRNAs were shown to be substrates for their cognate aminoacyl-tRNA synthetases. These findings demonstrate how nature can cope with the loss of the universal and supposedly ancient RNase P through genomic rearrangement at tRNA genes under the pressure of genome condensation.
Co-reporter:Benfang Ruan;Sotiria Palioura;Jeffrey Sabina;Laure Marvin-Guy;Sunil Kochhar;Robert A. LaRossa;Dieter Söll
PNAS 2008 Volume 105 (Issue 43 ) pp:16502-16507
Publication Date(Web):2008-10-28
DOI:10.1073/pnas.0809179105
A high level of accuracy during protein synthesis is considered essential for life. Aminoacyl-tRNA synthetases (aaRSs) translate
the genetic code by ensuring the correct pairing of amino acids with their cognate tRNAs. Because some aaRSs also produce
misacylated aminoacyl-tRNA (aa-tRNA) in vivo, we addressed the question of protein quality within the context of missense
suppression by Cys-tRNAPro, Ser-tRNAThr, Glu-tRNAGln, and Asp-tRNAAsn. Suppression of an active-site missense mutation leads to a mixture of inactive mutant protein (from translation with correctly
acylated aa-tRNA) and active enzyme indistinguishable from the wild-type protein (from translation with misacylated aa-tRNA).
Here, we provide genetic and biochemical evidence that under selective pressure, Escherichia coli not only tolerates the presence of misacylated aa-tRNA, but can even require it for growth. Furthermore, by using mass spectrometry
of a reporter protein not subject to selection, we show that E. coli can survive the ambiguous genetic code imposed by misacylated aa-tRNA tolerating up to 10% of mismade protein. The editing
function of aaRSs to hydrolyze misacylated aa-tRNA is not essential for survival, and the EF-Tu barrier against misacylated
aa-tRNA is not absolute. Rather, E. coli copes with mistranslation by triggering the heat shock response that stimulates nonoptimized polypeptides to achieve a native
conformation or to be degraded. In this way, E. coli ensures the presence of sufficient functional protein albeit at a considerable energetic cost.
Co-reporter:Mary Anne T. Rubio;Bethany Krett;Jesse J. Rinehart;Dieter Söll;Andreas S. Reichert;Stéphane Duvezin-Caubet;Juan D. Alfonzo
PNAS 2008 Volume 105 (Issue 27 ) pp:9186-9191
Publication Date(Web):2008-07-08
DOI:10.1073/pnas.0804283105
Mitochondrial genomes generally encode a minimal set of tRNAs necessary for protein synthesis. However, a number of eukaryotes
import tRNAs from the cytoplasm into their mitochondria. For instance, Saccharomyces cerevisiae imports cytoplasmic tRNAGln into the mitochondrion without any added protein factors. Here, we examine the existence of a similar active tRNA import
system in mammalian mitochondria. We have used subcellular RNA fractions from rat liver and human cells to perform RT-PCR
with oligonucleotide primers specific for nucleus-encoded tRNACUG
Gln and tRNAUUG
Gln species, and we show that these tRNAs are present in rat and human mitochondria in vivo. Import of in vitro transcribed tRNAs, but not of heterologous RNAs, into isolated mitochondria also demonstrates that this process is tRNA-specific
and does not require the addition of cytosolic factors. Although this in vitro system requires ATP, it is resistant to inhibitors of the mitochondrial electrochemical gradient, a key component of protein
import. tRNAGln import into mammalian mitochondria proceeds by a mechanism distinct from protein import. We also show that both tRNAGln species and a bacterial pre-tRNAAsp can be imported in vitro into mitochondria isolated from myoclonic epilepsy with ragged-red fiber cells if provided with sufficient ATP (2 mM). This
work suggests that tRNA import is more widespread than previously thought and may be a universal trait of mitochondria. Mutations
in mitochondrial tRNA genes have been associated with human disease; the tRNA import system described here could possibly
be exploited for the manipulation of defective mitochondria.
Co-reporter:Uttam L. RajBhandary;Dieter Söll
PNAS 2008 105 (14 ) pp:5285-5286
Publication Date(Web):2008-04-08
DOI:10.1073/pnas.0801193105
Co-reporter:Alexandre Ambrogelly;Sarath Gundllapalli;Stephanie Herring;Carla Polycarpo;Carina Frauer;Dieter Söll
PNAS 2007 Volume 104 (Issue 9 ) pp:3141-3146
Publication Date(Web):2007-02-27
DOI:10.1073/pnas.0611634104
Pyrrolysine (Pyl), the 22nd naturally encoded amino acid, gets acylated to its distinctive UAG suppressor tRNAPyl by the cognate pyrrolysyl-tRNA synthetase (PylRS). Here we determine the RNA elements required for recognition and aminoacylation
of tRNAPyl in vivo by using the Pyl analog N-ε-cyclopentyloxycarbonyl-l-lysine. Forty-two Methanosarcina barkeri tRNAPyl variants were tested in Escherichia coli for suppression of the lac amber A24 mutation; then relevant tRNAPyl mutants were selected to determine in vivo binding to M. barkeri PylRS in a yeast three-hybrid system and to measure in vitro tRNAPyl aminoacylation. tRNAPyl identity elements include the discriminator base, the first base pair of the acceptor stem, the T-stem base pair G51:C63,
and the anticodon flanking nucleotides U33 and A37. Transplantation of the tRNAPyl identity elements into the mitochondrial bovine tRNASer scaffold yielded chimeric tRNAs active both in vitro and in vivo. Because the anticodon is not important for PylRS recognition, a tRNAPyl variant could be constructed that efficiently suppressed the lac opal U4 mutation in E. coli. These data suggest that tRNAPyl variants may decode numerous codons and that tRNAPyl:PylRS is a fine orthogonal tRNA:synthetase pair that facilitated the late addition of Pyl to the genetic code.
Co-reporter:Jennifer M. Kavran;Sarath Gundllapalli;Patrick O'Donoghue;Markus Englert;Dieter Söll;Thomas A. Steitz;
Proceedings of the National Academy of Sciences 2007 104(27) pp:11268-11273
Publication Date(Web):June 25, 2007
DOI:10.1073/pnas.0704769104
Pyrrolysine (Pyl), the 22nd natural amino acid and genetically encoded by UAG, becomes attached to its cognate tRNA by pyrrolysyl-tRNA
synthetase (PylRS). We have determined three crystal structures of the Methanosarcina mazei PylRS complexed with either AMP–PNP, Pyl–AMP plus pyrophosphate, or the Pyl analogue N-ε-[(cylopentyloxy)carbonyl]-l-lysine plus ATP. The structures reveal that PylRS utilizes a deep hydrophobic pocket for recognition of the Pyl side chain.
A comparison of these structures with previously determined class II tRNA synthetase complexes illustrates that different
substrate specificities derive from changes in a small number of residues that form the substrate side-chain-binding pocket.
The knowledge of these structures allowed the placement of PylRS in the aminoacyl-tRNA synthetase (aaRS) tree as the last
known synthetase that evolved for genetic code expansion, as well as the finding that Pyl arose before the last universal
common ancestral state. The PylRS structure provides an excellent framework for designing new aaRSs with altered amino acid
specificity.
Co-reporter:Hiroyuki Oshikane;Kelly Sheppard;Shuya Fukai;Yuko Nakamura;Ryuichiro Ishitani;Tomoyuki Numata;R. Lynn Sherrer;Liang Feng;Emmanuelle Schmitt;Michel Panvert;Sylvain Blanquet;Yves Mechulam;Dieter Söll;Osamu Nureki
Science 2006 Vol 312(5782) pp:1950-1954
Publication Date(Web):30 Jun 2006
DOI:10.1126/science.1128470
Abstract
Glutaminyl–transfer RNA (Gln-tRNAGln) in archaea is synthesized in a pretranslational amidation of misacylated Glu-tRNAGln by the heterodimeric Glu-tRNAGln amidotransferase GatDE. Here we report the crystal structure of the Methanothermobacter thermautotrophicus GatDE complexed to tRNAGln at 3.15 angstroms resolution. Biochemical analysis of GatDE and of tRNAGln mutants characterized the catalytic centers for the enzyme's three reactions (glutaminase, kinase, and amidotransferase activity). A 40 angstrom–long channel for ammonia transport connects the active sites in GatD and GatE. tRNAGln recognition by indirect readout based on shape complementarity of the D loop suggests an early anticodon-independent RNA-based mechanism for adding glutamine to the genetic code.
Co-reporter:Dieter Söll;Hubert D. Becker;Pierre Plateau;Sylvain Blanquet;Michael Ibba
PNAS 2000 Volume 97 (Issue 26 ) pp:14224-14228
Publication Date(Web):2000-12-19
DOI:10.1073/pnas.97.26.14224
Lysyl-tRNA synthesis is catalyzed by two unrelated families of
aminoacyl-tRNA synthetases. In most bacteria and all eukarya, the known
lysyl-tRNA synthetases (LysRSs) are subclass IIb-type aminoacyl-tRNA
synthetases, whereas many archaea and a scattering of bacteria contain
an unrelated class I-type LysRS. Examination of the recognition of
partially modified tRNALys anticodon variants by a
bacterial (from Borrelia burgdorferi) and an archaeal
(from Methanococcus maripaludis) class I lysyl-tRNA
synthetase revealed differences in the pattern of anticodon recognition
between the two enzymes. U35 and U36 were both important for
recognition by the B. burgdorferi enzyme, whereas only
U36 played a role in recognition by M. maripaludis
LysRS. Examination of the phylogenetic distribution of class I LysRSs
suggested a correlation between recognition of U35 and U36 and the
presence of asparaginyl-tRNA synthetase (AsnRS), which also recognizes
U35 and U36 in the anticodon of tRNAAsn. However, the class
II LysRS of Helicobacter pylori, an organism that lacks
AsnRS, also recognizes both U35 and U36, indicating that the presence
of AsnRS has solely influenced the phylogenetic distribution of class I
LysRSs. These data suggest that competition between unrelated
aminoacyl-tRNA synthetases for overlapping anticodon sequences is a
determinant of the phylogenetic distribution of extant synthetase
families. Such patterns of competition also provide a basis for the two
separate horizontal gene transfer events hypothesized in the evolution
of the class I lysyl-tRNA synthetases.
Co-reporter:Corwin Miller, Markus J. Bröcker, Laure Prat, Kevan Ip, ... Dieter Söll
FEBS Letters (4 August 2015) Volume 589(Issue 17) pp:2194-2199
Publication Date(Web):4 August 2015
DOI:10.1016/j.febslet.2015.06.039
•A chimera of tRNASer and tRNASec, tRNAUTuX, binds EF-Tu to insert Sec at UAG codons.•tRNAUTuX was used for complete, high fidelity Sec insertion.•We show in vitro selenoprotein synthesis, compatible with wild-type and synthetic tRNA.•Sense codons were recoded in vitro in the presence of SelB.•Formate dehydrogenase activity demonstrates in vitro selenoenzyme synthesis.Incorporation of selenocysteine (Sec) in bacteria requires a UGA codon that is reassigned to Sec by the Sec-specific elongation factor SelB and a conserved mRNA motif (SECIS element). These requirements severely restrict the engineering of selenoproteins. Earlier, a synthetic tRNASec was reported that allowed canonical Sec incorporation by EF-Tu; however, serine misincorporation limited its scope. We report a superior tRNASec variant (tRNAUTuX) that facilitates EF-Tu dependent stoichiometric Sec insertion in response to UAG both in vivo in Escherichia coli and in vitro in a cellfree protein synthesis system. We also demonstrate recoding of several sense codons in a SelB supplemented cell-free system. These advances in Sec incorporation will aid rational design and directed evolution of selenoproteins.
Co-reporter:Kelly Sheppard, R. Lynn Sherrer, Dieter Söll
Journal of Molecular Biology (28 March 2008) Volume 377(Issue 3) pp:845-853
Publication Date(Web):28 March 2008
DOI:10.1016/j.jmb.2008.01.064
Many prokaryotes form the amide aminoacyl-tRNAs glutaminyl-tRNA and asparaginyl-tRNA by tRNA-dependent amidation of the mischarged tRNA species, glutamyl-tRNAGln or aspartyl-tRNAAsn. Archaea employ two such amidotransferases, GatCAB and GatDE, while bacteria possess only one, GatCAB. The Methanothermobacter thermautotrophicus GatDE is slightly more efficient using Asn as an amide donor than Gln (kcat/KM of 5.4 s−1/mM and 1.2 s−1/mM, respectively). Unlike the bacterial GatCAB enzymes studied to date, the M. thermautotrophicus GatCAB uses Asn almost as well as Gln as an amide donor (kcat/KM of 5.7 s−1/mM and 16.7 s−1/mM, respectively). In contrast to the initial characterization of the M. thermautotrophicus GatCAB as being able to form Asn-tRNAAsn and Gln-tRNAGln, our data demonstrate that while the enzyme is able to transamidate Asp-tRNAAsn (kcat/KM of 125 s−1/mM) it is unable to transamidate M. thermautotrophicus Glu-tRNAGln. However, M. thermautotrophicus GatCAB is capable of transamidating Glu-tRNAGln from H. pylori or B. subtilis, and M. thermautotrophicus Glu-tRNAAsn. Thus, M. thermautotrophicus encodes two amidotransferases, each with its own activity, GatDE for Gln-tRNA and GatCAB for Asn-tRNA synthesis.
Co-reporter:Kelly Sheppard, Dieter Söll
Journal of Molecular Biology (28 March 2008) Volume 377(Issue 3) pp:831-844
Publication Date(Web):28 March 2008
DOI:10.1016/j.jmb.2008.01.016
Glutaminyl-tRNA synthetase and asparaginyl-tRNA synthetase evolved from glutamyl-tRNA synthetase and aspartyl-tRNA synthetase, respectively, after the split in the last universal communal ancestor (LUCA). Glutaminyl-tRNAGln and asparaginyl-tRNAAsn were likely formed in LUCA by amidation of the mischarged species, glutamyl-tRNAGln and aspartyl-tRNAAsn, by tRNA-dependent amidotransferases, as is still the case in most bacteria and all known archaea. The amidotransferase GatCAB is found in both domains of life, while the heterodimeric amidotransferase GatDE is found only in Archaea. The GatB and GatE subunits belong to a unique protein family that includes Pet112 that is encoded in the nuclear genomes of numerous eukaryotes. GatE was thought to have evolved from GatB after the emergence of the modern lines of decent. Our phylogenetic analysis though places the split between GatE and GatB, prior to the phylogenetic divide between Bacteria and Archaea, and Pet112 to be of mitochondrial origin. In addition, GatD appears to have emerged prior to the bacterial–archaeal phylogenetic divide. Thus, while GatDE is an archaeal signature protein, it likely was present in LUCA together with GatCAB. Archaea retained both amidotransferases, while Bacteria emerged with only GatCAB. The presence of GatDE has favored a unique archaeal tRNAGln that may be preventing the acquisition of glutaminyl-tRNA synthetase in Archaea. Archaeal GatCAB, on the other hand, has not favored a distinct tRNAAsn, suggesting that tRNAAsn recognition is not a major barrier to the retention of asparaginyl-tRNA synthetase in many Archaea.
Co-reporter:Takuya Umehara, Jihyo Kim, Sangsik Lee, Li-Tao Guo, ... Hee-Sung Park
FEBS Letters (23 March 2012) Volume 586(Issue 6) pp:729-733
Publication Date(Web):23 March 2012
DOI:10.1016/j.febslet.2012.01.029
Posttranslational modifications play a crucial role in modulating protein structure and function. Genetic incorporation of unnatural amino acids into a specific site of a protein facilitates the systematic study of protein modifications including acetylation. We here report the directed evolution of pyrrolysyl-tRNA synthetase (PylRS) from Methanosarcina mazei to create N-acetyl lysyl-tRNA synthetases (AcKRSs) using a new selection system based on the killing activity of the toxic ccdB gene product. The amino acid specificity of these and of published [1,2] AckRSs was tested in vitro and in vivo, and the enzyme-kinetic properties of the AckRSs were evaluated for the first time.Highlights► N-acetyl lysyl-tRNA synthetases were evolved by a CcdB-based selection. ► N-acetyl lysine specificity was validated by both in vitro and in vivo approaches. ► Kinetic properties of the evolved synthetases were evaluated for the first time. ► The evolved synthetases will facilitate the systematic study of protein acetylation.
Co-reporter:Ilka U. Heinemann, Dieter Söll, Lennart Randau
FEBS Letters (21 January 2010) Volume 584(Issue 2) pp:303-309
Publication Date(Web):21 January 2010
DOI:10.1016/j.febslet.2009.10.067
Transfer RNA (tRNA) molecules are highly conserved in length, sequence and structure in order to be functional in the ribosome. However, mostly in archaea, the short genes encoding tRNAs can be found disrupted, fragmented, with permutations or with non-functional mutations of conserved nucleotides. Here, we give an overview of recently discovered tRNA maturation pathways that require intricate processing steps to finally generate the standard tRNA from these unusual tRNA genes.
Co-reporter:Patrick O’Donoghue, Laure Prat, Ilka U. Heinemann, Jiqiang Ling, ... Dieter Söll
FEBS Letters (2 November 2012) Volume 586(Issue 21) pp:3931-3937
Publication Date(Web):2 November 2012
DOI:10.1016/j.febslet.2012.09.033
Over 300 amino acids are found in proteins in nature, yet typically only 20 are genetically encoded. Reassigning stop codons and use of quadruplet codons emerged as the main avenues for genetically encoding non-canonical amino acids (NCAAs). Canonical aminoacyl-tRNAs with near-cognate anticodons also read these codons to some extent. This background suppression leads to ‘statistical protein’ that contains some natural amino acid(s) at a site intended for NCAA. We characterize near-cognate suppression of amber, opal and a quadruplet codon in common Escherichia coli laboratory strains and find that the PylRS/tRNAPyl orthogonal pair cannot completely outcompete contamination by natural amino acids.Highlights► Nonsense suppression by natural amino acids hinders genetic code expansion. ► Higher levels of near-cognate suppression found in opal versus amber codons. ► E. coli BL21 and MG1655 display higher background suppression than TOP10. ► PylRS/tRNAUCAPyl cannot outcompete Trp incorporation at opal codons. ► PylRS/tRNAUCCUPyl is outcompeted by Arg-tRNAArg in quadruplet decoding.
Co-reporter:Jing Yuan, Michael J. Hohn, R. Lynn Sherrer, Sotiria Palioura, ... Dieter Söll
FEBS Letters (2 July 2010) Volume 584(Issue 13) pp:2857-2861
Publication Date(Web):2 July 2010
DOI:10.1016/j.febslet.2010.05.028
The essential methanogen enzyme Sep-tRNA:Cys-tRNA synthase (SepCysS) converts O-phosphoseryl-tRNACys (Sep-tRNACys) into Cys-tRNACys in the presence of a sulfur donor. Likewise, Sep-tRNA:Sec-tRNA synthase converts O-phosphoseryl-tRNASec (Sep-tRNASec) to selenocysteinyl-tRNASec (Sec-tRNASec) using a selenium donor. While the Sep moiety of the aminoacyl-tRNA substrates is the same in both reactions, tRNACys and tRNASec differ greatly in sequence and structure. In an Escherichia coli genetic approach that tests for formate dehydrogenase activity in the absence of selenium donor we show that Sep-tRNASec is a substrate for SepCysS. Since Sec and Cys are the only active site amino acids known to sustain FDH activity, we conclude that SepCysS converts Sep-tRNASec to Cys-tRNASec, and that Sep is crucial for SepCysS recognition.
Co-reporter:Sunna Helgadóttir, Sylvie Sinapah, Dieter Söll, Jiqiang Ling
FEBS Letters (2 January 2012) Volume 586(Issue 1) pp:60-63
Publication Date(Web):2 January 2012
DOI:10.1016/j.febslet.2011.11.024
In methanogenic archaea, Sep-tRNA:Cys-tRNA synthase (SepCysS) converts Sep-tRNACys to Cys-tRNACys. The mechanism of tRNA-dependent cysteine formation remains unclear due to the lack of functional studies. In this work, we mutated 19 conserved residues in Methanocaldococcus jannaschii SepCysS, and employed an in vivo system to determine the activity of the resulting variants. Our results show that three active-site cysteines (Cys39, Cys42 and Cys247) are essential for SepCysS activity. In addition, combined with structural modeling, our mutational and functional analyses also reveal multiple residues that are important for the binding of PLP, Sep and tRNA. Our work thus represents the first systematic functional analysis of conserved residues in archaeal SepCysSs, providing insights into the catalytic and substrate binding mechanisms of this poorly characterized enzyme.Highlights► Mutational and functional studies identified 11 residues critical for SepCysS activity. ► All three active site cysteines of SepCysS are essential for tRNA-dependent Cys formation. ► The phosphate group of Sep is recognized by Arg79, His103 and Tyr104.
Co-reporter:Sarath Gundllapalli, Alexandre Ambrogelly, Takuya Umehara, Darrick Li, ... Dieter Söll
FEBS Letters (15 October 2008) Volume 582(Issues 23–24) pp:3353-3358
Publication Date(Web):15 October 2008
DOI:10.1016/j.febslet.2008.08.027
Methanosarcina barkeri inserts pyrrolysine (Pyl) at an in-frame UAG codon in its monomethylamine methyltransferase gene. Pyrrolysyl-tRNA synthetase acylates Pyl onto tRNAPyl, the amber suppressor pyrrolysine Pyl tRNA. Here we show that M. barkeri Fusaro tRNAPyl can be misacylated with serine by the M. barkeri bacterial-type seryl-tRNA synthetase in vitro and in vivo in Escherichia coli. Compared to the M. barkeri Fusaro tRNA, the M. barkeri MS tRNAPyl contains two base changes; a G3:U70 pair, the known identity element for E. coli alanyl-tRNA synthetase (AlaRS). While M. barkeri MS tRNAPyl cannot be alanylated by E. coli AlaRS, mutation of the MS tRNAPyl A4:U69 pair into C4:G69 allows aminoacylation by E. coli AlaRS both in vitro and in vivo.
Co-reporter:Jiqiang Ling, Chris Cho, Li-Tao Guo, Hans R. Aerni, ... Dieter Söll
Molecular Cell (14 December 2012) Volume 48(Issue 5) pp:713-722
Publication Date(Web):14 December 2012
DOI:10.1016/j.molcel.2012.10.001
Protein mistranslation causes growth arrest in bacteria, mitochondrial dysfunction in yeast, and neurodegeneration in mammals. It remains poorly understood how mistranslated proteins cause such cellular defects. Here we demonstrate that streptomycin, a bactericidal aminoglycoside that increases ribosomal mistranslation, induces transient protein aggregation in wild-type Escherichia coli. We further determined the aggregated proteome using label-free quantitative mass spectrometry. To identify genes that reduce cellular mistranslation toxicity, we selected from an overexpression library protein products that increased resistance against streptomycin and kanamycin. The selected proteins were significantly enriched in members of the oxidation-reduction pathway. Overexpressing one of these proteins, alkyl hydroperoxide reductase subunit F (a protein defending bacteria against hydrogen peroxide), but not its inactive mutant suppressed aggregated protein formation upon streptomycin treatment and increased aminoglycoside resistance. This work provides in-depth analyses of an aggregated proteome caused by streptomycin and suggests that cellular defense against hydrogen peroxide lowers the toxicity of mistranslation.Graphical AbstractDownload high-res image (251KB)Download full-size imageHighlights► In-depth coverage of the aggregated proteome induced by streptomycin in E. coli ► Identified proteins are susceptible to oxidation and streptomycin-induced aggregation ► Alkyl hydroperoxide reductase suppresses protein aggregation caused by streptomycin ► Oxidation-reduction proteins increase bacterial resistance against aminoglycosides
Co-reporter:Markus Englert, Sarath Moses, Michael Hohn, Jiqiang Ling, ... Dieter Söll
FEBS Letters (11 October 2013) Volume 587(Issue 20) pp:3360-3364
Publication Date(Web):11 October 2013
DOI:10.1016/j.febslet.2013.08.037
•O-Phosphoseryl-tRNA synthetase aminoacylates the 2′-OH of tRNA terminal adenosine.•Pyrrolysyl-tRNA synthetase aminoacylates the 3′-OH of tRNA terminal adenosine.•O-phosphoseryl-tRNA synthetase resembles phenylalanyl-tRNA synthetase.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.
Co-reporter:Stephanie Herring, Alexandre Ambrogelly, Sarath Gundllapalli, Patrick O’Donoghue, ... Dieter Söll
FEBS Letters (10 July 2007) Volume 581(Issue 17) pp:3197-3203
Publication Date(Web):10 July 2007
DOI:10.1016/j.febslet.2007.06.004
Pyrrolysine (Pyl) is co-translationally inserted into a subset of proteins in the Methanosarcinaceae and in Desulfitobacterium hafniense programmed by an in-frame UAG stop codon. Suppression of this UAG codon is mediated by the Pyl amber suppressor tRNA, tRNAPyl, which is aminoacylated with Pyl by pyrrolysyl-tRNA synthetase (PylRS). We compared the behavior of several archaeal and bacterial PylRS enzymes towards tRNAPyl. Equilibrium binding analysis revealed that archaeal PylRS proteins bind tRNAPyl with higher affinity (KD = 0.1–1.0 μM) than D. hafniense PylRS (KD = 5.3–6.9 μM). In aminoacylation the archaeal PylRS enzymes did not distinguish between archaeal and bacterial tRNAPyl species, while the bacterial PylRS displays a clear preference for the homologous cognate tRNA. We also show that the amino-terminal extension present in archaeal PylRSs is dispensable for in vitro activity, but required for PylRS function in vivo.
Co-reporter:Jae-hyeong Ko, Yane-Shih Wang, Akiyoshi Nakamura, Li-Tao Guo, ... Takuya Umehara
FEBS Letters (1 October 2013) Volume 587(Issue 19) pp:3243-3248
Publication Date(Web):1 October 2013
DOI:10.1016/j.febslet.2013.08.018
•Molecular evolution of PylRS reveals the ancestral PheRS activity.•Superfolder GFP reporter enables rapid analysis of PylRS substrate specificity.•The pocket size is critical to Phe recognition and rejection of Phe analogs.Pyrrolysyl-tRNA synthetase (PylRS) is a class IIc aminoacyl-tRNA synthetase that is related to phenylalanyl-tRNA synthetase (PheRS). Genetic selection provided PylRS variants with a broad range of specificity for diverse non-canonical amino acids (ncAAs). One variant is a specific phenylalanine-incorporating enzyme. Structural models of the PylRSamino acid complex show that the small pocket size and π-interaction play an important role in specific recognition of Phe and the engineered PylRS active site resembles that of Escherichia coli PheRS.
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