Motor Output Variability in Movement Disorders: Insights From Essential Tremor.

Findings on individuals with essential tremor suggest that tremor (within-trial movement unsteadiness) and inconsistency (trial-to-trial movement variance) stem from distinct pathologies and affect function uniquely. Nonetheless, the intricacies of inconsistency in movement disorders remain largely unexplored, as exemplified in ataxia where inconsistency below healthy levels is associated with greater pathology. We advocate for clinical assessments that quantify both tremor and inconsistency.

Adolescent boys who participate in sports exhibit similar ramp torque control with young men despite differences in strength and tendon characteristics.

PURPOSE: The goal of this paper was to determine if sports participation influences torque control differently for adolescent boys and young men during a slow ramp task. METHODS: Twenty-one adolescent boys (11 athletes) and 31 young men (16 athletes) performed a slow ramp increase in plantar flexion torque from 0 to maximum. We quantified torque control as the coefficient of variation (CV) of torque during the ramp and quantified the Achilles tendon mechanical properties using ultrasonography. RESULTS: Relative to adolescent boys, young men were taller, heavier, stronger, and had a longer and stiffer Achilles tendon. However, these characteristics were not different between athletes and non-athletes in adolescent boys. For the CV of torque, there was a significant interaction with sports participation, indicating that only adolescent boys who were non-athletes had greater variability than young men. The CV of torque of all participants was predicted from the maximum torque and torque oscillations from 1 to 2 Hz, whereas the CV of torque for adolescent boys was predicted only from torque oscillations from 1 to 2 Hz. CONCLUSION: These findings suggested that adolescent boys who participate in sports exhibited lower torque variability during a slow ramp task, which was not explained by differences in Achilles tendon properties or strength.

Temporal Invariance in SCA6 Is Related to Smaller Cerebellar Lobule VI and Greater Disease Severity.

Regulating muscle force and timing are fundamental for accurate motor performance. In spinocerebellar ataxia type 6 (SCA6), there is evidence that individuals have greater force dysmetria but display better temporal accuracy during fast goal directed contractions. Here, we test whether greater temporal accuracy occurs in all individuals with SCA6, and can be explained by lesser temporal variability. Further we examine whether it is linked to disease severity and specific degenerative changes in the cerebellum. Nineteen human participants with SCA6 (13 woman) and 18 healthy controls performed fast goal-directed ankle dorsiflexion contractions aiming at a spatiotemporal target. We quantified the endpoint control of these contractions, gray matter (GM) integrity of the cerebellum, and disease severity using the International Cooperative Ataxia Rating Scale (ICARS). SCA6 individuals exhibited lower temporal endpoint error and variability than the healthy controls (p = 0.008). Statistically, SCA6 clustered into two distinct groups for temporal variability. A group with low temporal variability ranging from 10 to 19% (SCA6a) and a group with temporal variability similar to healthy controls (SCA6b; 19-40%).SCA6a exhibited greater disease severity than SCA6b, as assessed with ICARS (p < 0.001). Lower temporal variability, which was not associated with disease duration (R2 = 0.1, p > 0.2), did correlate with both greater ICARS (R2 = 0.3) and reduced GM volume in cerebellar lobule VI (R2 = 0.35). Other cerebellar lobules did not relate to temporal variability. We provide new evidence that a subset of SCA6 with greater loss of GM in cerebellum lobule VI exhibit temporal invariance and more severe ataxia than other SCA6 individuals.SIGNIFICANCE STATEMENT Variability is an inherent feature of voluntary movement, and traditionally more variability in the targeted output infers impaired performance. For example, cerebellar patients present exacerbated temporal variability during multijoint movements, which is thought to contribute to their motor deficits. In the current work, we show that in a subgroup of spinocerebellar ataxia type 6 individuals, temporal variability is lower than that of healthy controls when performing single-joint fast-goal directed movements. This invariance related to exacerbated atrophy of lobule VI of the cerebellum and exacerbated disease severity. The relation between invariance and disease severity suggests that pathological motor variability can manifest not only as an exacerbation but also as a reduction relative to healthy controls.

Quantitative Separation of Tremor and Ataxia in Essential Tremor.

OBJECTIVE: This study addresses an important problem in neurology, distinguishing tremor and ataxia using quantitative methods. Specifically, we aimed to quantitatively separate dysmetria, a cardinal sign of ataxia, from tremor in essential tremor (ET). METHODS: In Experiment 1, we compared 19 participants diagnosed with ET undergoing thalamic deep brain stimulation (DBS; ETDBS ) to 19 healthy controls (HC). We quantified tremor during postural tasks using accelerometry and dysmetria with fast, reverse-at-target goal-directed movements. To ensure that endpoint accuracy was unaffected by tremor, we quantified dysmetria in selected trials manifesting a smooth trajectory to the endpoint. Finally, we manipulated tremor amplitude by switching DBS ON and OFF to examine its effect on dysmetria. In Experiment 2, we compared 10 ET participants with 10 HC to determine whether we could identify and distinguish dysmetria from tremor in non-DBS ET. RESULTS: Three findings suggest that we can quantify dysmetria independently of tremor in ET. First, ETDBS and ET exhibited greater dysmetria than HC and dysmetria did not correlate with tremor (R2 < 0.01). Second, even for trials with tremor-free trajectories to the target, ET exhibited greater dysmetria than HC (p < 0.01). Third, activating DBS reduced tremor (p < 0.01) but had no effect on dysmetria (p > 0.2). INTERPRETATION: We demonstrate that dysmetria can be quantified independently of tremor using fast, reverse-at-target goal-directed movements. These results have important implications for the understanding of ET and other cerebellar and tremor disorders. Future research should examine the neurophysiological mechanisms underlying each symptom and characterize their independent contribution to disability. ANN NEUROL 2020;88:375-387.

Biosynthesis and function of posttranscriptional modifications of transfer RNAs.

Posttranscriptional modifications of transfer RNAs (tRNAs) are critical for all core aspects of tRNA function, such as folding, stability, and decoding. Most tRNA modifications were discovered in the 1970s; however, the near-complete description of the genes required to introduce the full set of modifications in both yeast and Escherichia coli is very recent. This led to a new appreciation of the key roles of tRNA modifications and tRNA modification enzymes as checkpoints for tRNA integrity and for integrating translation with other cellular functions such as transcription, primary metabolism, and stress resistance. A global survey of tRNA modification enzymes shows that the functional constraints that drive the presence of modifications are often conserved, but the solutions used to fulfill these constraints differ among different kingdoms, organisms, and species.

Mutations in PROSC Disrupt Cellular Pyridoxal Phosphate Homeostasis and Cause Vitamin-B6-Dependent Epilepsy.

Pyridoxal 5'-phosphate (PLP), the active form of vitamin B6, functions as a cofactor in humans for more than 140 enzymes, many of which are involved in neurotransmitter synthesis and degradation. A deficiency of PLP can present, therefore, as seizures and other symptoms that are treatable with PLP and/or pyridoxine. Deficiency of PLP in the brain can be caused by inborn errors affecting B6 vitamer metabolism or by inactivation of PLP, which can occur when compounds accumulate as a result of inborn errors of other pathways or when small molecules are ingested. Whole-exome sequencing of two children from a consanguineous family with pyridoxine-dependent epilepsy revealed a homozygous nonsense mutation in proline synthetase co-transcribed homolog (bacterial), PROSC, which encodes a PLP-binding protein of hitherto unknown function. Subsequent sequencing of 29 unrelated indivduals with pyridoxine-responsive epilepsy identified four additional children with biallelic PROSC mutations. Pre-treatment cerebrospinal fluid samples showed low PLP concentrations and evidence of reduced activity of PLP-dependent enzymes. However, cultured fibroblasts showed excessive PLP accumulation. An E.coli mutant lacking the PROSC homolog (ΔYggS) is pyridoxine sensitive; complementation with human PROSC restored growth whereas hPROSC encoding p.Leu175Pro, p.Arg241Gln, and p.Ser78Ter did not. PLP, a highly reactive aldehyde, poses a problem for cells, which is how to supply enough PLP for apoenzymes while maintaining free PLP concentrations low enough to avoid unwanted reactions with other important cellular nucleophiles. Although the mechanism involved is not fully understood, our studies suggest that PROSC is involved in intracellular homeostatic regulation of PLP, supplying this cofactor to apoenzymes while minimizing any toxic side reactions.

Motor planning perturbation: muscle activation and reaction time.

Reaction time (RT) is the time interval between the appearance of a stimulus and initiation of a motor response. Within RT, two processes occur, selection of motor goals and motor planning. An unresolved question is whether perturbation to the motor planning component of RT slows the response and alters the voluntary activation of muscle. The purpose of this study was to determine how the modulation of muscle activity during an RT response changes with motor plan perturbation. Twenty-four young adults (20.5 ±1.1 yr, 13 women) performed 15 trials of an isometric RT task with ankle dorsiflexion using a sinusoidal anticipatory strategy (10-20% maximum voluntary contraction). We compared the processing part of the RT and modulation of muscle activity from 10 to 60 Hz of the tibialis anterior (primary agonist) when the stimulus appeared at the trough or at the peak of the sinusoidal task. We found that RT ( P = 0.003) was longer when the stimulus occurred at the peak compared with the trough. During the time of the reaction, the electromyography (EMG) power from 10 to 35 Hz was less at the peak than the trough ( P = 0.019), whereas the EMG power from 35 to 60 Hz was similar between the peak and trough ( P = 0.92). These results suggest that perturbation to motor planning lengthens the processing part of RT and alters the voluntary activation of the muscle by decreasing the relative amount of power from 10 to 35 Hz. NEW & NOTEWORTHY We aimed to determine whether perturbation to motor planning would alter the speed and muscle activity of the response. We compared trials when a stimulus appeared at the peak or trough of an oscillatory reaction time task. When the stimulus occurred at the trough, participants responded faster, with greater force, and less EMG power from 10-35 Hz. We provide evidence that motor planning perturbation slows the response and alters the voluntary activity of the muscle.

Speed but not amplitude of visual feedback exacerbates force variability in older adults.

Magnification of visual feedback (VF) impairs force control in older adults. In this study, we aimed to determine whether the age-associated increase in force variability with magnification of visual feedback is a consequence of increased amplitude or speed of visual feedback. Seventeen young and 18 older adults performed a constant isometric force task with the index finger at 5% of MVC. We manipulated the vertical (force gain) and horizontal (time gain) aspect of the visual feedback so participants performed the task with the following VF conditions: (1) high amplitude-fast speed; (2) low amplitude-slow speed; (3) high amplitude-slow speed. Changing the visual feedback from low amplitude-slow speed to high amplitude-fast speed increased force variability in older adults but decreased it in young adults (P < 0.01). Changing the visual feedback from low amplitude-slow speed to high amplitude-slow speed did not alter force variability in older adults (P > 0.2), but decreased it in young adults (P < 0.01). Changing the visual feedback from high amplitude-slow speed to high amplitude-fast speed increased force variability in older adults (P < 0.01) but did not alter force variability in young adults (P > 0.2). In summary, increased force variability in older adults with magnification of visual feedback was evident only when the speed of visual feedback increased. Thus, we conclude that in older adults deficits in the rate of processing visual information and not deficits in the processing of more visual information impair force control.

Motor plan differs for young and older adults during similar movements.

Older adults exhibit altered activation of the agonist and antagonist muscles during goal-directed movements compared with young adults. However, it remains unclear whether the differential activation of the antagonistic muscles in older adults results from an impaired motor plan or an altered ability of the muscle to contract. The purpose of this study, therefore, was to determine whether the motor plan differs for young and older adults. Ten young (26.1 ± 4.3 yr, 4 women) and 16 older adults (71.9 ± 6.9 yr, 9 women) participated in the study. Participants performed 100 trials of fast goal directed movements with ankle dorsiflexion while we recorded the electromyographic activity of the primary agonist (tibialis anterior; TA) and antagonist (soleus; SOL) muscles. From those 100 trials we selected 5 trials in each of 3 movement end-point categories (fast, accurate, and slow). We investigated age-associated differences in the motor plan by quantifying the individual activity and coordination of the agonist and antagonist muscles. During similar movement end points, older adults exhibited similar activation of the agonist (TA) and antagonist (SOL) muscles compared with young adults. In addition, the coordination of the agonist and antagonist muscles (TA and SOL) was different between the two age groups. Specifically, older adults exhibited lower TA-SOL overlap (F1,23 = 41.2, P < 0.001) and greater TA-SOL peak EMG delay (F1,25 = 35.5, P < 0.001). This finding suggests that although subjects in both age groups displayed similar movement end points, they exhibited a different motor plan, as demonstrated by altered coordination between the agonist and antagonist muscles.NEW & NOTEWORTHY We aimed to determine whether the altered activation of muscles in older adults compared with young adults during fast goal-directed movements is related to an altered motor plan. For matched movements, there were differences in the coordination of antagonistic muscles but no differences in the individual activation of muscles. We provide novel evidence that the differential activation of muscles in older adults is related to an altered motor plan.

Voluntary reduction of force variability via modulation of low-frequency oscillations.

Visual feedback can influence the force output by changing the power in frequencies below 1 Hz. However, it remains unknown whether visual guidance can help an individual reduce force variability voluntarily. The purpose of this study, therefore, was to determine whether an individual can voluntarily reduce force variability during constant contractions with visual guidance, and whether this reduction is associated with a decrease in the power of low-frequency oscillations (0-1 Hz) in force and muscle activity. Twenty young adults (27.6 ± 3.4 years) matched a force target of 15% MVC (maximal voluntary contraction) with ankle dorsiflexion. Participants performed six visually unrestricted contractions, from which we selected the trial with the least variability. Following, participants performed six visually guided contractions and were encouraged to reduce their force variability within two guidelines (±1 SD of the least variable unrestricted trial). Participants decreased the SD of force by 45% (P < 0.001) during the guided condition, without changing mean force (P > 0.2). The decrease in force variability was associated with decreased low-frequency oscillations (0-1 Hz) in force (R 2 = 0.59), which was associated with decreased low-frequency oscillations in EMG bursts (R 2 = 0.35). The reduction in low-frequency oscillations in EMG burst was positively associated with power in the interference EMG from 35 to 60 Hz (R 2 = 0.47). In conclusion, voluntary reduction of force variability is associated with decreased low-frequency oscillations in EMG bursts and consequently force output. We provide novel evidence that visual guidance allows healthy young adults to reduce force variability voluntarily likely by adjusting the low-frequency oscillations in the neural drive.

Sex differences in spatial accuracy relate to the neural activation of antagonistic muscles in young adults.

Sex is an important physiological variable of behavior, but its effect on motor control remains poorly understood. Some evidence suggests that women exhibit greater variability during constant contractions and poorer accuracy during goal-directed tasks. However, it remains unclear whether motor output variability or altered muscle activation impairs accuracy in women. Here, we examine sex differences in endpoint accuracy during ankle goal-directed movements and the activity of the antagonistic muscles. Ten women (23.1 ± 5.1 years) and 10 men (23 ± 3.7 years) aimed to match a target (9° in 180 ms) with ankle dorsiflexion. Participants performed 50 trials and we recorded the endpoint accuracy and the electromyographic (EMG) activity of the primary agonist (Tibialis Anterior; TA) and antagonist (Soleus; SOL) muscles. Women exhibited greater spatial inaccuracy (Position error: t = -2.65, P = 0.016) but not temporal inaccuracy relative to men. The motor output variability was similar for the two sexes (P > 0.2). The spatial inaccuracy in women was related to greater variability in the coordination of the antagonistic muscles (R 2 0.19, P = 0.03). These findings suggest that women are spatially less accurate than men during fast goal-directed movements likely due to an altered activation of the antagonistic muscles.

High-throughput comparison, functional annotation, and metabolic modeling of plant genomes using the PlantSEED resource.

The increasing number of sequenced plant genomes is placing new demands on the methods applied to analyze, annotate, and model these genomes. Today's annotation pipelines result in inconsistent gene assignments that complicate comparative analyses and prevent efficient construction of metabolic models. To overcome these problems, we have developed the PlantSEED, an integrated, metabolism-centric database to support subsystems-based annotation and metabolic model reconstruction for plant genomes. PlantSEED combines SEED subsystems technology, first developed for microbial genomes, with refined protein families and biochemical data to assign fully consistent functional annotations to orthologous genes, particularly those encoding primary metabolic pathways. Seamless integration with its parent, the prokaryotic SEED database, makes PlantSEED a unique environment for cross-kingdom comparative analysis of plant and bacterial genomes. The consistent annotations imposed by PlantSEED permit rapid reconstruction and modeling of primary metabolism for all plant genomes in the database. This feature opens the unique possibility of model-based assessment of the completeness and accuracy of gene annotation and thus allows computational identification of genes and pathways that are restricted to certain genomes or need better curation. We demonstrate the PlantSEED system by producing consistent annotations for 10 reference genomes. We also produce a functioning metabolic model for each genome, gapfilling to identify missing annotations and proposing gene candidates for missing annotations. Models are built around an extended biomass composition representing the most comprehensive published to date. To our knowledge, our models are the first to be published for seven of the genomes analyzed.

Essentiality of threonylcarbamoyladenosine (t(6)A), a universal tRNA modification, in bacteria.

Threonylcarbamoyladenosine (t(6)A) is a modified nucleoside universally conserved in tRNAs in all three kingdoms of life. The recently discovered genes for t(6)A synthesis, including tsaC and tsaD, are essential in model prokaryotes but not essential in yeast. These genes had been identified as antibacterial targets even before their functions were known. However, the molecular basis for this prokaryotic-specific essentiality has remained a mystery. Here, we show that t(6)A is a strong positive determinant for aminoacylation of tRNA by bacterial-type but not by eukaryotic-type isoleucyl-tRNA synthetases and might also be a determinant for the essential enzyme tRNA(Ile)-lysidine synthetase. We confirm that t(6)A is essential in Escherichia coli and a survey of genome-wide essentiality studies shows that genes for t(6)A synthesis are essential in most prokaryotes. This essentiality phenotype is not universal in Bacteria as t(6)A is dispensable in Deinococcus radiodurans, Thermus thermophilus, Synechocystis PCC6803 and Streptococcus mutans. Proteomic analysis of t(6)A(-) D. radiodurans strains revealed an induction of the proteotoxic stress response and identified genes whose translation is most affected by the absence of t(6)A in tRNAs. Thus, although t(6)A is universally conserved in tRNAs, its role in translation might vary greatly between organisms.

Evidence that COG0325 proteins are involved in PLP homeostasis.

Pyridoxal 5'-phosphate (PLP) is an essential cofactor for nearly 60 Escherichia coli enzymes but is a highly reactive molecule that is toxic in its free form. How PLP levels are regulated and how PLP is delivered to target enzymes are still open questions. The COG0325 protein family belongs to the fold-type III class of PLP enzymes and binds PLP but has no known biochemical activity although it occurs in all kingdoms of life. Various pleiotropic phenotypes of the E. coli COG0325 (yggS) mutant have been reported, some of which were reproduced and extended in this study. Comparative genomic, genetic and metabolic analyses suggest that these phenotypes reflect an imbalance in PLP homeostasis. The E. coli yggS mutant accumulates the PLP precursor pyridoxine 5'-phosphate (PNP) and is sensitive to an excess of pyridoxine but not of pyridoxal. The pyridoxine toxicity phenotype is complemented by the expression of eukaryotic yggS orthologs. It is also suppressed by the presence of amino acids, specifically isoleucine, threonine and leucine, suggesting the PLP-dependent enzyme transaminase B (IlvE) is affected. These genetic results lay a foundation for future biochemical studies of the role of COG0325 proteins in PLP homeostasis.

The flavoprotein Mcap0476 (RlmFO) catalyzes m5U1939 modification in Mycoplasma capricolum 23S rRNA.

Efficient protein synthesis in all organisms requires the post-transcriptional methylation of specific ribosomal ribonucleic acid (rRNA) and transfer RNA (tRNA) nucleotides. The methylation reactions are almost invariably catalyzed by enzymes that use S-adenosylmethionine (AdoMet) as the methyl group donor. One noteworthy exception is seen in some bacteria, where the conserved tRNA methylation at m5U54 is added by the enzyme TrmFO using flavin adenine dinucleotide together with N5,N10-methylenetetrahydrofolate as the one-carbon donor. The minimalist bacterium Mycoplasma capricolum possesses two homologs of trmFO, but surprisingly lacks the m5U54 tRNA modification. We created single and dual deletions of the trmFO homologs using a novel synthetic biology approach. Subsequent analysis of the M. capricolum RNAs by mass spectrometry shows that the TrmFO homolog encoded by Mcap0476 specifically modifies m5U1939 in 23S rRNA, a conserved methylation catalyzed by AdoMet-dependent enzymes in all other characterized bacteria. The Mcap0476 methyltransferase (renamed RlmFO) represents the first folate-dependent flavoprotein seen to modify ribosomal RNA.

A role for the universal Kae1/Qri7/YgjD (COG0533) family in tRNA modification.

The YgjD/Kae1 family (COG0533) has been on the top-10 list of universally conserved proteins of unknown function for over 5 years. It has been linked to DNA maintenance in bacteria and mitochondria and transcription regulation and telomere homeostasis in eukaryotes, but its actual function has never been found. Based on a comparative genomic and structural analysis, we predicted this family was involved in the biosynthesis of N(6)-threonylcarbamoyl adenosine, a universal modification found at position 37 of tRNAs decoding ANN codons. This was confirmed as a yeast mutant lacking Kae1 is devoid of t(6)A. t(6)A(-) strains were also used to reveal that t(6)A has a critical role in initiation codon restriction to AUG and in restricting frameshifting at tandem ANN codons. We also showed that YaeZ, a YgjD paralog, is required for YgjD function in vivo in bacteria. This work lays the foundation for understanding the pleiotropic role of this universal protein family.

Cross kingdom functional conservation of the core universally conserved threonylcarbamoyladenosine tRNA synthesis enzymes.

Threonylcarbamoyladenosine (t(6)A) is a universal modification located in the anticodon stem-loop of tRNAs. In yeast, both cytoplasmic and mitochondrial tRNAs are modified. The cytoplasmic t(6)A synthesis pathway was elucidated and requires Sua5p, Kae1p, and four other KEOPS complex proteins. Recent in vitro work suggested that the mitochondrial t(6)A machinery of Saccharomyces cerevisiae is composed of only two proteins, Sua5p and Qri7p, a member of the Kae1p/TsaD family (L. C. K. Wan et al., Nucleic Acids Res. 41:6332-6346, 2013, http://dx.doi.org/10.1093/nar/gkt322). Sua5p catalyzes the first step leading to the threonyl-carbamoyl-AMP intermediate (TC-AMP), while Qri7 transfers the threonyl-carbamoyl moiety from TC-AMP to tRNA to form t(6)A. Qri7p localizes to the mitochondria, but Sua5p was reported to be cytoplasmic. We show that Sua5p is targeted to both the cytoplasm and the mitochondria through the use of alternative start sites. The import of Sua5p into the mitochondria is required for this organelle to be functional, since the TC-AMP intermediate produced by Sua5p in the cytoplasm is not transported into the mitochondria in sufficient amounts. This minimal t(6)A pathway was characterized in vitro and, for the first time, in vivo by heterologous complementation studies in Escherichia coli. The data revealed a potential for TC-AMP channeling in the t(6)A pathway, as the coexpression of Qri7p and Sua5p is required to complement the essentiality of the E. coli tsaD mutant. Our results firmly established that Qri7p and Sua5p constitute the mitochondrial pathway for the biosynthesis of t(6)A and bring additional advancement in our understanding of the reaction mechanism.

Unique Neural Mechanisms Underlying Speed Control of Low-Force Ballistic Contractions.

According to the speed-control hypothesis, the rate of force development (RFD) during ballistic contractions is dictated by force amplitude because time to peak force (TPF) remains constant regardless of changes in force amplitude. However, this hypothesis has not been tested at force levels below 20% of an individual's maximum voluntary contraction (MVC). Here, we examined the relationship between the RFD and force amplitude from 2 to 85% MVC and the underlying structure of muscle activity in 18 young adults. Participants exerted ballistic index finger abductions for 50 trials in each of seven randomly assigned force levels (2, 5, 15, 30, 50, 70, and 85% MVC). We quantified TPF, RFD, and various EMG burst characteristics. Contrary to the speed-control hypothesis, we found that TPF was not constant, but significantly varied from 2 to 85% MVC. Specifically, the RFD slope from 2 to 15% MVC was greater than the RFD slope from 30 to 85% MVC. Longer TPF at low force levels was associated with the variability of EMG burst duration, whereas longer TPF with higher force levels was associated with the EMG burst integral. Contrary to the speed-control hypothesis, we found that the regulation of TPF for low and high force levels was different, suggesting that neuronal variability is critical for force levels below 30% MVC and neuronal amplitude for force levels above 30% MVC. These findings present compelling new evidence highlighting the limitations of the speed-control hypothesis underscoring the need for a new theoretical framework.

Biosynthesis of threonylcarbamoyl adenosine (t6A), a universal tRNA nucleoside.

The anticodon stem-loop (ASL) of transfer RNAs (tRNAs) drives decoding by interacting directly with the mRNA through codon/anticodon pairing. Chemically complex nucleoside modifications found in the ASL at positions 34 or 37 are known to be required for accurate decoding. Although over 100 distinct modifications have been structurally characterized in tRNAs, only a few are universally conserved, among them threonylcarbamoyl adenosine (t(6)A), found at position 37 in the anticodon loop of a subset of tRNA. Structural studies predict an important role for t(6)A in translational fidelity, and in vivo work supports this prediction. Although pioneering work in the 1970s identified the fundamental substrates for t(6)A biosynthesis, the enzymes responsible for its biosynthesis have remained an enigma. We report here the discovery that in bacteria four proteins (YgjD, YrdC, YjeE, and YeaZ) are both necessary and sufficient for t(6)A biosynthesis in vitro. Notably, YrdC and YgjD are members of universally conserved families that were ranked among the top 10 proteins of unknown function in need of functional characterization, while YeaZ and YjeE are specific to bacteria. This latter observation, coupled with the essentiality of all four proteins in bacteria, establishes this pathway as a compelling new target for antimicrobial development.

Gcn4 misregulation reveals a direct role for the evolutionary conserved EKC/KEOPS in the t6A modification of tRNAs.

The EKC/KEOPS complex is universally conserved in Archaea and Eukarya and has been implicated in several cellular processes, including transcription, telomere homeostasis and genomic instability. However, the molecular function of the complex has remained elusive so far. We analyzed the transcriptome of EKC/KEOPS mutants and observed a specific profile that is highly enriched in targets of the Gcn4p transcriptional activator. GCN4 expression was found to be activated at the translational level in mutants via the defective recognition of the inhibitory upstream ORFs (uORFs) present in its leader. We show that EKC/KEOPS mutants are defective for the N6-threonylcarbamoyl adenosine modification at position 37 (t(6)A(37)) of tRNAs decoding ANN codons, which affects initiation at the inhibitory uORFs and provokes Gcn4 de-repression. Structural modeling reveals similarities between Kae1 and bacterial enzymes involved in carbamoylation reactions analogous to t(6)A(37) formation, supporting a direct role for the EKC in tRNA modification. These findings are further supported by strong genetic interactions of EKC mutants with a translation initiation factor and with threonine biosynthesis genes. Overall, our data provide a novel twist to understanding the primary function of the EKC/KEOPS and its impact on several essential cellular functions like transcription and telomere homeostasis.