Raffael Schaffrath

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Verified

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• PhD
• Professor (Full) at University of Kassel

Transfer RNA (tRNA) molecules are essential to decode messenger RNA codons during protein synthesis. All known tRNAs are heavily modified at multiple positions through post-transcriptional addition of chemical groups. Modifications in the tRNA anticodons are directly influencing ribosome decoding and dynamics during translation elongation and are c...

Previously, combined loss of different anticodon loop modifications was shown to impair the function of distinct tRNAs in Saccharomyces cerevisiae. Surprisingly , each scenario resulted in shared cellular phenotypes, the basis of which is unclear. Since loss of tRNA modification may evoke transcriptional responses , we characterized global transcri...

tRNA modifications affect ribosomal elongation speed and co-translational folding dynamics. The Elongator complex is responsible for introducing 5-carboxymethyl at wobble uridine bases (cm⁵U34) in eukaryotic tRNAs. However, the structure and function of human Elongator remain poorly understood. In this study, we present a series of cryo-EM structur...

Post-translational modifications by ubiquitin-like proteins (UBLs) are essential for nearly all cellular processes. Ubiquitin-related modifier 1 (Urm1) is a unique UBL, which plays a key role in tRNA anticodon thiolation as a sulfur carrier protein (SCP) and is linked to the noncanonical E1 enzyme Uba4 (ubiquitin-like protein activator 4). While Ur...

Diphthamide, a post-translationally modified histidine residue of eukaryotic TRANSLATION ELONGATION FACTOR2 (eEF2), is the human host cell-sensitizing target of diphtheria toxin. Diphthamide biosynthesis depends on the 4Fe-4S-cluster protein Dph1 catalyzing the first committed step, as well as Dph2 to Dph7, in yeast and mammals. Here we show that d...

The diphthamide modification of eukaryotic translation elongation factor (eEF2) is important for accurate protein synthesis. While the enzymes for diphthamide synthesis are known, coordination of eEF2 synthesis with the diphthamide modification to maintain only modified eEF2 is unknown. Physical and genetic interactions extracted from BioGRID show...

Diphthamide is a post-translationally modified histidine residue of eukaryotic TRANSLATION ELONGATION FACTOR 2 (eEF2) and the target of diphtheria toxin in human cells. In yeast and mammals, the 4Fe-4S cluster-containing proteins Dph1 and Dph2 catalyze the first biosynthetic step of diphthamide formation. Here we identify Arabidopsis thaliana DPH2...

The Dph1•Dph2 heterodimer from yeast is a radical SAM (RS) enzyme that generates the 3-amino-3-carboxy-propyl (ACP) precursor for diphthamide, a clinically relevant modification on eukaryotic elongation factor 2 (eEF2). ACP formation requires SAM cleavage and atypical Cys-bound Fe-S clusters in each Dph1 and Dph2 subunit. Intriguingly, the first Cy...

The Dph1•Dph2 heterodimer from yeast is a radical SAM (RS) enzyme that generates the 3-amino-3-carboxy-propyl (ACP) precursor for diphthamide, a clinically relevant modification on eukaryotic elongation factor 2 (eEF2). ACP formation requires SAM cleavage and atypical Cys-bound Fe-S clusters in each Dph1 and Dph2 subunit. Intriguingly, the first Cy...

Therapeutic fluoropyrimidines 5-fluorouracil (5-FU) and 5-fluorocytosine (5-FC) are in long use for treatment of human cancers and severe invasive fungal infections, respectively. 5-Fluorouridine triphosphate represents a bioactive metabolite of both drugs and is incorporated into target cells’ RNA. Here we use the model fungus Saccharomyces cerevi...

The Elongator complex plays a pivotal role in the wobble uridine modification of the tRNA anticodon. Comprising two sets of six distinct subunits, namely, Elongator proteins (ELP1-ELP6) and associated proteins, the holo-Elongator complex demonstrates remarkable functional and structural conservation across eukaryotes. However, the precise details o...

The Dph1•Dph2 heterodimer from yeast is a radical SAM (RS) enzyme that generates the 3-amino-3-carboxy-propyl (ACP) precursor for diphthamide, a clinically relevant modification on eukaryotic elongation factor 2 (eEF2). ACP formation requires SAM cleavage and atypical Cys-bound Fe-S clusters in each Dph1 and Dph2 subunit. Intriguingly, the first Cy...

The Dph1•Dph2 heterodimer from yeast is a radical SAM (RS) enzyme that generates the 3-amino-3-carboxy-propyl (ACP) precursor for diphthamide, a clinically relevant modification on eukaryotic elongation factor 2 (eEF2). ACP formation requires SAM cleavage and atypical Cys-bound Fe-S clusters in each Dph1 and Dph2 subunit. Intriguingly, the first Cy...

In the new article series in Trends in Molecular Medicine, 'Science around the world', we embark on a journey to learn about thebscientific background and expertise of the authors and the geographic landscape that has shaped their groundbreaking work. We have compiled a list of questions to highlight their experience at a specific location, and we...

Diphthamide, a complex modification on eukaryotic translation elongation factor 2 (eEF2), assures reading-frame fidelity during translation. Diphthamide and enzymes for its synthesis are conserved in eukaryotes and archaea. Originally identified as target for diphtheria toxin (DT) in humans, its clinical relevance now proves to be broader than the...

Guided by evidence from archaeal orthologues, we searched for a putative SAM-binding pocket in Dph1•Dph2 from Saccharomyces cerevisiae. We predict an SAM-binding pocket near the FeS cluster domain that is conserved across eukaryotes in Dph1 but not Dph2. Site-directed DPH1 mutagenesis and functional characterization through assay diagnostics for th...

In eukaryotes, the Dph1•Dph2 dimer is a non-canonical radical SAM enzyme. Using iron-sulfur (FeS) clusters, it cleaves the cosubstrate S-adenosyl-methionine (SAM) to form a 3-amino-3-carboxy-propyl (ACP) radical for the synthesis of diphthamide. The latter decorates a histidine residue on elongation factor 2 (EF2) conserved from archaea to yeast an...

The chemical biology of native nucleic acid modifications has seen an intense upswing, first concerning DNA modifications in the field of epigenetics and then concerning RNA modifications in a field that was correspondingly rebaptized epitranscriptomics by analogy. The German Research Foundation (DFG) has funded several consortia with a scientific...

In eukaryotes, the Dph1•Dph2 dimer is a non-canonical radical SAM enzyme. Using iron-sulfur (FeS) clusters, it cleaves the cosubstrate S-adenosyl-methionine (SAM) to form a 3-amino-3-carboxy-propyl (ACP) radical for synthesis of diphthamide. The latter decorates a histidine residue on elongation factor 2 (EF2) conserved from archaea to yeast and hu...

In the yeast Saccharomyces cerevisiae, the absence of the pseudouridine synthase Pus3/Deg1, which modifies tRNA positions 38 and 39, results in increased lipid droplet (LD) content and translational defects. In addition, starvation‐like transcriptome alterations and induced protein aggregation were observed. In this study, we show that the deg1 mut...

The autosomal recessive diphthamide deficiency syndrome presents as intellectual disability with developmental abnormalities, seizures, craniofacial and additional morphological phenotypes. It is caused by reduced activity of proteins that synthesize diphthamide on human translation elongation factor 2. Diphthamide synthesis requires seven proteins...

In yeast, Elongator-dependent tRNA modifications are regulated by the Kti11•Kti13 dimer and hijacked for cell killing by zymocin, a tRNase ribotoxin. Kti11 (alias Dph3) also controls modification of elongation factor 2 (EF2) with diphthamide, the target for lethal ADP-ribosylation by diphtheria toxin (DT). Diphthamide formation on EF2 involves four...

Radical S-adenosyl-methionine (RS) enzymes use iron-sulfur (FeS) clusters and often modify biological macromolecules (i.e., lipids, proteins, nucleic acids). One such unique protein modification known as diphthamide is conserved from yeast to human cells. It decorates elongation factor 2 (EF2), which helps decode genetic information to make new pro...

Posttranslational modifications by ubiquitin-like proteins (UBLs) are essential cellular processes. Ubiquitin-related modifier 1 (Urm 1) is an ancient UBL, involved in tRNA anticodon thiolation as a sulfur carrier protein (SCP). While Urm 1 has also been observed to conjugate to proteins like other UBLs, the molecular mechanism underlying its coval...

The Elongator complex in eukaryotes has conserved tRNA modification functions and contributes to various physiological processes such as transcriptional control, DNA replication and repair, and chromatin accessibility. ARABIDOPSIS ELONGATOR PROTEIN 4 (AtELP4) is one of the six subunits (AtELP1–AtELP6) in Arabidopsis Elongator. In addition, there is...

Purpose: Diphthamide is a post-translationally modified histidine essential for messenger RNA translation and ribosomal protein synthesis. We present evidence for DPH5 as a novel cause of embryonic lethality and profound neurodevelopmental delays (NDDs). Methods: Molecular testing was performed using exome or genome sequencing. A targeted Dph5 knoc...

Post-translational modifications by ubiquitin-like proteins (UBLs) are essential for nearly all cellular processes. Ubiquitin-related modifier 1 (Urm1) is a unique UBL, which plays a key role in tRNA anticodon thiolation as a sulfur carrier protein (SCP) and is linked to the noncanonical E1 enzyme Uba4 (ubiquitin-like protein activator 4). While Ur...

Purpose: Diphthamide is a post-translationally modified histidine essential for messenger RNA translation and ribosomal protein synthesis. We present evidence for DPH5 as a novel cause of embryonic lethality and profound neurodevelopmental delays (NDDs). Methods: Molecular testing was performed using exome or genome sequencing. A targeted Dph5 kno...

Protein disulfide isomerases (PDIs) function in forming the correct disulfide bonds in client proteins, thereby aiding the folding of proteins that enter the secretory pathway. Recently, several PDIs have been identified as targets of organic electrophiles, yet the client proteins of specific PDIs remain largely undefined. Here, we report that PDIs...

Study purpose: Neurodevelopmental disorders (NDDs) are genetically heterogeneous lifelong conditions with a known etiology in approximately 50% of individuals. Here, we report DPH5 (Diphthamide biosynthesis protein 5) as a novel cause of embryonic lethality, multisystem dysfunction and profound NDDs in three unrelated families.

Ubiquitin related modifier 1 (Urm1) is a unique eukaryotic member of the ubiquitin-fold (UbF) protein family and conserved from yeast to humans. Urm1 is dual-functional, acting both as a sulfur carrier for thiolation of tRNA anticodons and as a protein modifier in a lysine-directed Ub-like conjugation also known as urmylation. Although Urm1 conjuga...

In 2000, Nobel Prize laureate Professor Yoshinori Ohsumi discovered a unique ubiquitin-like protein in yeast. Urm1 (ubiquitin related modifier 1) is dual functional and acts as a protein and tRNA modifier. Interestingly, both functions require the initial sulfur transfer onto Urm1, generating a thio-activated intermediate. However, such a sulfur de...

Yeast phenotypes associated with the lack of wobble uridine (U34) modifications in tRNA were shown to be modulated by an allelic variation of SSD1, a gene encoding an mRNA-binding protein. We demonstrate that phenotypes caused by the loss of Deg1-dependent tRNA pseudouridylation are similarly affected by SSD1 allelic status.

Yeast phenotypes associated with the lack of wobble uridine (U34) modifications in tRNA were shown to be modulated by an allelic variation of SSD1, a gene encoding an mRNA-binding protein. We demonstrate that phenotypes caused by the loss of Deg1-dependent tRNA pseudouridylation are similarly affected by SSD1 allelic status. Temperature sensitivity...

In eukaryotes, Kti12 enables carboxymethylation (cm5) of transfer ribonucleic acids (tRNA) by the Elongator complex. The modification is located inside the anticodon of tRNA and supports correct translation via improved base pairing during decoding of messenger RNA (mRNA). Interestingly, either down or upregulation of Elongator have been associated...

Using microbial models (bacteria, yeast/fungi), the research of the Schaffrath laboratory has been focussing on organismic interaction between microbes and cell proliferation control in response to changes in environmental and/or intracellular cues. In doing so, they have provided innovative insights into both the physiological and pathobiological...

Posttranscriptional modifications of anticodon loops contribute to the decoding efficiency of tRNAs by supporting codon recognition and loop stability. Consistently, strong synthetic growth defects are observed in yeast strains simultaneously lacking distinct anticodon loop modifications. These phenotypes are accompanied by translational inefficien...

In the Elongator-dependent modification pathway, chemical modifications are introduced at the wobble uridines at position 34 in transfer RNAs (tRNAs), which serve to optimize codon translation rates. Here, we show that this three-step modification pathway exists in Dictyostelium discoideum, model of the evolutionary superfamily Amoebozoa. Not only...

In the Elongator-dependent modification pathway, chemical modifications are introduced at the wobble uridines at position 34 in transfer RNAs (tRNAs), which serve to optimize codon translation rates. Here, we show that this three-step modification pathway exists in Dictyostelium discoideum, model of the evolutionary superfamily Amoebozoa. Not only...

We describe a novel type of ribosomopathy that is defined by deficiency in diphthamidylation of translation elongation factor 2. The ribosomopathy was identified by correlating phenotypes and biochemical properties of previously described patients with diphthamide biosynthesis gene 1 (DPH1) deficiencies with a new patient that carried inactivating...

We describe a novel type of ribosomopathy that is defined by deficiency in diphthamidylation of translation elongation factor 2. The ribosomopathy was identified by correlating phenotypes and biochemical properties of previously described patients with diphthamide biosynthesis gene 1 (DPH1) deficiencies with a new patient that carried inactivating...

Modifications found in the Anticodon Stem Loop (ASL) of tRNAs play important roles in regulating translational speed and accuracy. Threonylcarbamoyl adenosine (t6A37) and 5-methoxycarbonyl methyl-2-thiouridine (mcm5s2U34) are critical ASL modifications that have been linked to several human diseases. The model yeast Saccharomyces cerevisiae is viab...

The yeast peroxiredoxin Ahp1undergoes urmylation, a lysine-directed conjugation to ubiquitin-like modifier Urm1. Although urmylation coincides with oxidative stress, it is unclear how this modification happens on a molecular level and whether it affects peroxiredoxin activity. Here, we report that thioredoxin mutants decrease Ahp1 urmylation in yea...

The yeast peroxiredoxin Ahp1, like related anti-oxidant enzymes in other species, undergoes urmylation, a lysine-directed conjugation to ubiquitin-like modifier Urm1. Ahp1 assembles into a homodimer that detoxifies peroxides via forming intersubunit disulfides between peroxidatic and resolving cysteines that are subsequently reduced by the thioredo...

Modifications found in the Anticodon Stem Loop (ASL) of tRNAs play important roles in regulating translational speed and accuracy. Threonylcarbamoyl adenosine (t6A37) and 5-methoxycarbonylmethyl-thiouridine (mcm5s2U34) are critical ASL modifications that have been linked to several human diseases. The model yeast Saccharomyces cerevisiae is viable...

The bifunctional ubiquitin related modifier 1 (Urm1), a well conserved ubiquitin-like protein, acts as a sulfur donor for tRNA thiolation and as a protein modifier for urmylation. Among Urm1 targets from baker’s yeast is Ahp1, a 2-Cys peroxiredoxin, which detoxifies ROS using redox-active thiols in resolving (CR31) and peroxidatic (CP62) cysteins....

Transfer RNAs (tRNAs) function as adapter molecules to transport appropriate amino acids to translating ribosomes according to mRNA codons, which are decoded by the tRNA anticodons. Moreover, tRNAs are subject to chemical alterations of the standard RNA nucleosides including methylations, thiolations, base isomerizations and complex side chain addi...

Posttranscriptional RNA modifications occur in all domains of life. Modifications of anticodon bases are of particular importance for ribosomal decoding and proteome homeostasis. The Elongator complex modifies uridines in the wobble position and is highly conserved in eukaryotes. Despite recent insights into Elongator's architecture, the structure...

Accurate quantification of the copy numbers of noncoding RNA has recently emerged as an urgent problem, with impact on fields such as RNA modification research, tissue differentiation, and others. Herein, we present a hybridization‐based approach that uses microscale thermophoresis (MST) as a very fast and highly precise readout to quantify, for ex...

Posttranscriptional RNA modifications occur in all domains of life. Modifications of anticodon bases are of particular importance for ribosomal decoding and proteome homeostasis. The Elongator complex modifies uridines in the wobble position and is highly conserved in eukaryotes. Despite recent insights into Elongator's architecture, the structure...

Die biologische Uhr tickt. Alles Lebendige altert. Vom Einzeller bis zum Elefanten, alle Lebewesen sind diesem Naturgesetz unterworfen. Das Vorrücken der Uhrzeiger kann niemand beeinflussen, weder stoppen noch beschleunigen. Oder? Nicht ganz. Bei kleinsten Lebewesen sieht es anders aus. Die Mitarbeiter der Fachgebiete Mikrobiologie und Makromolekul...

Accurate quantification of copy numbers of noncoding RNA has recently emerged as an urgent problem, with impact on fields such as RNA modification research, tissue differentiation, and others. Here we present a hybridization‐based approach that uses microscale thermophoresis (MST) as a very fast and highly precise readout to quantify e.g. single tR...

Accurate quantification of copy numbers of noncoding RNA has recently emerged as an urgent problem, with impact on fields such as RNA modification research, tissue differentiation, and others. Here we present a hybridization‐based approach that uses microscale thermophoresis (MST) as a very fast and highly precise readout to quantify e.g. single tR...

The dual functional ubiquitin related modifier 1 (Urm1), a well conserved ubiquitin-like protein, acts as a protein modifier via so called urmylation and as a sulfur donor for tRNA thiolation. Using the 2-Cys peroxiredoxin Ahp1 capable of detoxifying reactive oxygen species (ROS) as a bona fide Urm1 target, we examined urmylation requirements and c...

Transfer RNA (tRNA) is subject to a multitude of posttranscriptional modifications which can profoundly impact its functionality as the essential adaptor molecule in messenger RNA (mRNA) translation. Therefore, dynamic regulation of tRNA modification in response to environmental changes can tune the efficiency of gene expression in concert with the...

Modifications in the anticodon loop of transfer RNAs (tRNAs) have been shown to ensure optimal codon translation rates and prevent protein homeostasis defects that arise in response to translational pausing. Consequently, several yeast mutants lacking important anticodon loop modifications were shown to accumulate protein aggregates. Here we analyz...

In eukaryotes, the modification of an invariant histidine (His-699 in yeast) residue in translation elongation factor 2 (EF2) with diphthamide involves a conserved pathway encoded by the DPH1-DPH7 gene network. Diphthamide is the target for diphtheria toxin and related lethal ADP ribosylases, which collectively kill cells by inactivating the essent...

SGA data for DPH1-DPH7 and EFT1-EFT2 query genes based on TheCellMap.org database mining. (XLS)

Negative interactions between EF2 and diphthamide gene deletions in composite mutant (dph2Δeft2Δ) reduce growth and doubling times. Shown are growth curves of wild-type (wt), single (dph2Δ and eft2Δ) and double mutant (dph2Δ eft2Δ) cells monitored over a period of 23 h in liquid rich (YPD) medium. Cells were cultivated in single batches of 50 ml me...

Yeast strains used or generated for this study. (DOCX)

Primers used in this study. (DOCX)

Synthetic sick growth phenotypes result from combining EF2 downregulation with loss of diphthamide. Ten-fold serial cell dilutions of wild-type (wt), single (dph4Δ and eft2Δ) and composite (dph4Δ eft2Δ) mutants were cultivated at different temperatures (30°C, 37°C, 39°C or 40°C) or incubated in the absence (untreated) or presence of various chemica...

Hygromycin phenotypes of mutants lacking diphthamide and/or proper EF2 supply. Serial cell dilutions of the indicated yeast stains were cultivated on media without (untreated) or supplemented with various hygromycin doses (40, 80 μg/ml) at 30°C for 2–3 days. (TIF)

Diphthamide is a posttranslational histidine modification of translation elongation factor 2 (EF2). It’s name refers to the lethal inactivation of EF2 upon ADP ribosylation by diphtheria toxin, which targets diphthamide and inhibits ribosomal translocation. Although this emphasizes a pathological role, precisely why cells need to make diphthamide,...

Certain tRNA species contain Elongator dependent modifications at anticodon wobble uridines (U34). These are targets for zymocin, a fungal ribotoxin produced by Kluyveromyces lactis. Zymocin cleaves in the anticodon of modified tRNAs and thereby results in yeast cell death. Mutations in the Elongator complex result in loss of U34 modification and r...

Transfer RNA (tRNA) from all domains of life contains multiple modified nucleosides, the functions of which remain incompletely understood. Genetic interactions between tRNA modification genes in Saccharomyces cerevisiae suggest that different tRNA modifications collaborate to maintain translational efficiency. Here we characterize such collaborati...

Transfer RNAs are post-transcriptionally modified at multiple positions to ensure stability and decoding capacity. Many tRNA modifications in yeast are dispensable per se but their simultaneous absence can be highly deleterious for growth and stress resistance and induces shared cytological abnormalities. We used a combined transcriptomics and prot...

The protein phosphatase Sit4 has been shown to be required for lipogenesis and resistance against the acetyl-CoA carboxylase inhibitor soraphen A. Since Sit4 is also required for biosynthesis of Elongator dependent tRNA modifications such as 5-methoxycarbonylmethyluridine (mcm 5 U), we investigated the relevance of tRNA modifications in lipogenesis...

The Division of Microbiology led by Professor Schaffrath uses the budding yeast Saccharomyces cerevisiae as a model to study protein and RNA modification pathways that influence mRNA translation, protein synthesis and cell growth. One of the protein modifications the group is interested in is the so-called urmylation. Here, the protein Urm1 is tran...

The genetic alphabet consists of the four letters: C, A, G, and T in DNA and C,A,G, and U in RNA. Triplets of these four letters jointly encode 20 different amino acids out of which proteins of all organisms are built. This system is universal and is found in all kingdoms of life. However, bases in DNA and RNA can be chemically modified. In DNA, ar...

The secretion of antimicrobial proteins termed killer toxins is common among yeast species from diverse phylogenetic origin. Killer toxin-encoding genes may be localized either in the nucleus or on cytoplasmic double-stranded DNA or RNA molecules of viral ancestry. A number of distinct strategies are utilized by killer toxins to inhibit or kill com...

Elucidating the biology of yeast in its full complexity has major implications for science, medicine and industry. One of the most critical processes determining yeast life and physiology is cellular demise. However, the investigation of yeast cell death is a relatively young field, and a widely accepted set of concepts and terms is still missing....

Simultaneous removal of critical tRNA modifications such as 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U), pseudouridine 38/39 (ᴪ38/39) or cyclic threonyl-carbamoyladenosine (ct6A37) induces synthetic negative phenotypes in yeast. We characterized proteomic and transcriptomic changes in mutant combinations previously shown to affect either tRNALy...

Elucidating the biology of yeast in its full complexity has major implications for science, medicine and industry. One of the most critical processes determining yeast life and physiology is cellular demise. However, the investigation of yeast cell death is a relatively young field, and a widely accepted set of concepts and terms is still missing....

Ribonucleotide modifications perform a wide variety of roles in synthesis, turnover and functionality of tRNA molecules. The presence of particular chemical moieties can refine the internal interaction network within a tRNA molecule, influence its thermodynamic stability, contribute novel chemical properties and affect its decoding behavior during...

The Division of Microbiology at Kassel University uses Saccharomyces cerevisiae, a well-established yeast model for eukaryotic cell biology, to study protein and tRNA modification pathways that influence mRNA translation and cell growth. Recently, the group's research into iron-sulfur (FeS) cluster containing radical enzymes and related requirement...

In yeast, the nonsense suppressor tRNATyr (SUP4) decodes ochre stop codons due to a G to U exchange at the wobble position, generating an UAA cognate anticodon. To study the impact of tRNA modifications in the anticodon stem loop and variable region on SUP4 function, we quantified ochre read-through using a dual luciferase reporter assay. Absence o...

Saccharomyces cerevisiae cells are killed by zymocin, a tRNase ribotoxin complex from Kluyveromyces lactis, which cleaves anticodons and inhibits protein synthesis. Zymocin's action requires specific chemical modification of uridine bases in the anticodon wobble position (U34) by the Elongator complex (Elp1-Elp6). Hence, loss of anticodon modificat...

Saccharomyces cerevisiae cells are killed by zymocin, a tRNase ribotoxin complex from Kluyveromyces lactis, which cleaves anticodons and inhibits protein synthesis. Zymocin’s action requires specific chemical modification of uridine bases in the anticodon wobble position (U34) by the Elongator complex (Elp1-Elp6). Hence, loss of anticodon modificat...