UUG Codon Research Articles

Taurine for Mitochondrial Diseases

Mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) syndrome is characterized by a mitochondrial DNA mutation that leads to defective taurine modification of the leucine tRNA anticodon, with consequent impaired translation of the UUG codon. This defect reduces synthesis of respiratory chain complexes, which causes energy failure. Taurine supplementation improved mitochondrial function in MELAS model cells. A physician-initiated clinical trial reported that high-dose taurine supplementation therapy suppressed stroke-like episodes and improved taurine modification rates in leukocytes.

Altered proteome in translation initiation fidelity defective eIF5G31R mutant causes oxidative stress and DNA damage

The recognition of the AUG start codon and selection of an open reading frame (ORF) is fundamental to protein biosynthesis. Defect in the fidelity of start codon selection adversely affect proteome and have a pleiotropic effect on cellular function. Using proteomic techniques, we identified differential protein abundance in the translation initiation fidelity defective eIF5G31R mutant that initiates translation using UUG codon in addition to the AUG start codon. Consistently, the eIF5G31R mutant altered proteome involved in protein catabolism, nucleotide biosynthesis, lipid biosynthesis, carbohydrate metabolism, oxidation–reduction pathway, autophagy and re-programs the cellular pathways. The utilization of the upstream UUG codons by the eIF5G31R mutation caused downregulation of uridylate kinase expression, sensitivity to hydroxyurea, and DNA damage. The eIF5G31R mutant cells showed lower glutathione levels, high ROS activity, and sensitivity to H2O2.

US5/Rps2 residues at the 40S ribosome entry channel enhance initiation at suboptimal start codons in vivo.

The eukaryotic 43S pre-initiation complex (PIC) containing Met-tRNAiMet in a ternary complex (TC) with eIF2-GTP scans the mRNA leader for an AUG codon in favorable "Kozak" context. AUG recognition triggers rearrangement of the PIC from an open conformation to a closed state with more tightly bound Met-tRNAiMet. Yeast ribosomal protein uS5/Rps2 is located at the mRNA entry channel of the 40S subunit in the vicinity of mRNA nucleotides downstream from the AUG codon or rRNA residues that communicate with the decoding center, but its participation in start codon recognition was unknown. We found that nonlethal substitutions of conserved Rps2 residues in the entry channel reduce bulk translation initiation and increase discrimination against poor initiation codons. A subset of these substitutions suppress initiation at near-cognate UUG start codons in a yeast mutant with elevated UUG initiation, and also increase discrimination against AUG codons in suboptimal Kozak context, thus resembling previously described substitutions in uS3/Rps3 at the 40S entry channel or initiation factors eIF1 and eIF1A. In contrast, other Rps2 substitutions selectively discriminate against either near-cognate UUG codons, or poor Kozak context of an AUG or UUG start codon. These findings suggest that different Rps2 residues are involved in distinct mechanisms involved in discriminating against different features of poor initiation sites in vivo.

EIF2α interactions with mRNA control accurate start codon selection by the translation preinitiation complex.

In translation initiation, AUG recognition triggers rearrangement of the 48S preinitiation complex (PIC) from an open conformation to a closed state with more tightly-bound Met-tRNAi. Cryo-EM structures have revealed interactions unique to the closed complex between arginines R55/R57 of eIF2α with mRNA, including the -3 nucleotide of the 'Kozak' context. We found that R55/R57 substitutions reduced recognition of a UUG start codon at HIS4 in Sui- cells (Ssu- phenotype); and in vitro, R55G-R57E accelerated dissociation of the eIF2·GTP·Met-tRNAi ternary complex (TC) from reconstituted PICs with a UUG start codon, indicating destabilization of the closed complex. R55/R57 substitutions also decreased usage of poor-context AUGs in SUI1 and GCN4 mRNAs in vivo. In contrast, eIF2α-R53 interacts with the rRNA backbone only in the open complex, and the R53E substitution enhanced initiation at a UUG codon (Sui- phenotype) and poor-context AUGs, while reducing the rate of TC loading (Gcd- phenotype) in vivo. Consistently, R53E slowed TC binding to the PIC while decreasing TC dissociation at UUG codons in vitro, indicating destabilization of the open complex. Thus, distinct interactions of eIF2α with rRNA or mRNA stabilize first the open, and then closed, conformation of the PIC to influence the accuracy of initiation in vivo.

Translation Initiation Site Profiling Reveals Widespread Synthesis of Non-AUG-Initiated Protein Isoforms in Yeast.

Genomic analyses in budding yeast have helped define the foundational principles of eukaryotic gene expression. However, in the absence of empirical methods for defining coding regions, these analyses have historically excluded specific classes of possible coding regions, such as those initiating at non-AUG start codons. Here, we applied an experimental approach to globally annotate translation initiation sites in yeast and identified 149 genes with alternative N-terminally extended protein isoforms initiating from near-cognate codons upstream of annotated AUG start codons. These isoforms are produced in concert with canonical isoforms and translated with high specificity, resulting from initiation at only a small subset of possible start codons. The non-AUG initiation driving their production is enriched during meiosis and induced by low eIF5A, which is seen in this context. These findings reveal widespread production of non-canonical protein isoforms and unexpected complexity to the rules by which even a simple eukaryotic genome is decoded.

EIF1 discriminates against suboptimal initiation sites to prevent excessive uORF translation genome-wide.

The translation preinitiation complex (PIC) scans the mRNA for an AUG codon in a favorable context. Previous findings suggest that the factor eIF1 discriminates against non-AUG start codons by impeding full accommodation of Met-tRNAi in the P site of the 40S ribosomal subunit, necessitating eIF1 dissociation for start codon selection. Consistent with this, yeast eIF1 substitutions that weaken its binding to the PIC increase initiation at UUG codons on a mutant his4 mRNA and particular synthetic mRNA reporters; and also at the AUG start codon of the mRNA for eIF1 itself owing to its poor Kozak context. It was not known however whether such eIF1 mutants increase initiation at suboptimal start codons genome-wide. By ribosome profiling, we show that the eIF1-L96P variant confers increased translation of numerous upstream open reading frames (uORFs) initiating with either near-cognate codons (NCCs) or AUGs in poor context. The increased uORF translation is frequently associated with the reduced translation of the downstream main coding sequences (CDS). Initiation is also elevated at certain NCCs initiating amino-terminal extensions, including those that direct mitochondrial localization of the GRS1 and ALA1 products, and at a small set of main CDS AUG codons with especially poor context, including that of eIF1 itself. Thus, eIF1 acts throughout the yeast translatome to discriminate against NCC start codons and AUGs in poor context; and impairing this function enhances the repressive effects of uORFs on CDS translation and alters the ratios of protein isoforms translated from near-cognate versus AUG start codons.

Emerging Variants of the Integrative and Conjugant Element ICEMh1 in Livestock Pathogens: Structural Insights, Potential Host Range, and Implications for Bacterial Fitness and Antimicrobial Therapy.

Horizontal gene transfer of integrative and conjugative elements (ICE) in bacterial pathogens of the bovine respiratory disease (BRD) complex has emerged as a significant cause of antimicrobial resistance (AMR) and therapeutic failure and mortalities in cattle. The aim of this study was to assess an AMR ICE occurring in Pasteurella multocida from a case of BRD, designated ICEMh1PM22 for its structure and host genome insertion site, and to identify consequences for host fitness and antimicrobial therapy. The modular structure of ICEMh1-like elements found in several related livestock pathogens was compared to ICEMh1PM22, and the repertoire of cargo genes in variable ICE modules was functionally categorized. AMR genes were identified as frequent additions to the variable modules of ICEMh1-like elements. Random PCR-based mapping of ICEMh1PM22-genome junctions in transconjugants provided evidence that ICEMh1PM22 integrates into the tRNA-leu for the UUG codon, and not into tRNA-leu for other codons. This was separately confirmed in the genomes of ICEMh1-like-harboring livestock pathogens. Bacterial genera harboring receptive tRNA-leuUUG were identified to establish the potential host range of ICEMh1-like elements. ICEMh1PM22-carrying transconjugants in P. multocida and Mannheimia haemolytica were less fit than isogenic strains without the ICE when grown without antimicrobial selection. This fitness cost was abrogated in the presence of subinhibitory concentrations of antimicrobials. Despite this cost, ICEMh1PM22 was retained in transconjugants in extended culture. To identify possible therapeutic efficiencies, antimicrobial combinations were screened for synergistic interactions against AMR ICEMh1PM22-carrying transconjugants. No antimicrobial combination tested exhibited synergistic interactions against AMR P. multocida or M. haemolytica harboring ICEMh1PM22. In conclusion, this study provided information on the structural variation of ICEMh1-like elements, refined the ICE insertion site and potential host range, and demonstrated the risk and consequences for AMR following horizontal transfer of ICE into BRD pathogens.

Alternative Translation Initiation at a UUG Codon Gives Rise to Two Functional Variants of the Mitochondrial Protein Kgd4

Kgd4 is a novel subunit of the mitochondrial α-ketoglutarate dehydrogenase complex (KGDH). In yeast, the protein is present in two forms of unknown origin, as there is only one open reading frame and no alternative splicing. Here, we show that the two forms of Kgd4 derive from one mRNA that is translated by employing two alternative start sites. The standard, annotated AUG codon gives rise to the short form of the protein, while an upstream UUG codon is utilized to generate the larger form. However, both forms can be efficiently imported into mitochondria and stably incorporate into KGDH to support its activity. Translation of the long variant depends on sequences directly upstream of the alternative initiation site, demonstrating that translation initiation and its efficiency are dictated by the sequence context surrounding a specific codon. In summary, the two forms of Kgd4 follow a very unusual biogenesis pathway, supporting the notion that translation initiation in yeast is more flexible than it is widely recognized.

A network of eIF2β interactions with eIF1 and Met-tRNAi promotes accurate start codon selection by the translation preinitiation complex.

In translation initiation, a 43S preinitiation complex (PIC) containing eIF1 and a ternary complex (TC) of GTP-bound eIF2 and Met-RNAi scans the mRNA for the start codon. AUG recognition triggers eIF1 release and rearrangement from an open PIC conformation to a closed state with more tightly-bound Met-tRNAi (PIN state). Cryo-EM models reveal eIF2β contacts with eIF1 and Met-tRNAi exclusive to the open complex that should destabilize the closed state. eIF2β or eIF1 substitutions disrupting these contacts increase initiation at UUG codons, and compound substitutions also derepress translation of GCN4, indicating slower TC recruitment. The latter substitutions slow TC loading while stabilizing TC binding at UUG codons in reconstituted PICs, indicating a destabilized open complex and shift to the closed/PIN state. An eIF1 substitution that should strengthen the eIF2β:eIF1 interface has the opposite genetic and biochemical phenotypes. eIF2β is also predicted to restrict Met-tRNAi movement into the closed/PIN state, and substitutions that should diminish this clash increase UUG initiation in vivo and stabilize Met-tRNAi binding at UUG codons in vitro with little effect on TC loading. Thus, eIF2β anchors eIF1 and TC to the open complex, enhancing PIC assembly and scanning, while impeding rearrangement to the closed conformation at non-AUG codons.

Fidelity of HIS4 start codon selection influences 3-amino-1,2,4-triazole sensitivity in GTPase activating protein function defective eIF5

The eIF5 protein plays an important role in the fidelity of AUG start codon selection. However, the hyper GTPase eIF5G31R mutation in yeast causes preferential utilization of UUG as initiation codon and is termed as suppressor of initiation codon (Sui-) phenotype. The eIF5G31R mutant recognizes upUUG initiation codon from the 5' regulatory leader region of GCN4 transcript and dominantly represses GCN4 expression thereby conferring sensitivity to 3-amino-1,2,4-triazole (3AT)-induced starvation. The 3AT sensitivity was rescued by supplementing HIS4UUG allele. The eIF5G31R mutant has a better efficiency of UUG codon recognition from the HIS4UUG allele under starvation conditions. Moreover, the expression of HIS4UUG allele was significantly lower than the critical level causing additional derepression of GCN4 expression in eIF5G31R mutant to rescue its 3AT sensitivity. The overexpression of eIF1 improved expression of HIS4AUG allele and GCN4 transcript causing 3AT resistance, whereas overexpression of eIF1 resulted in diminished UUG codon recognition of HIS4UUG allele causing 3AT sensitivity, despite having higher GCN4 expression. This paper reports the critical role of HIS4 expression necessary in response to 3AT-induced starvation in the eIF5G31R mutant which is ostensibly not a direct target of 3AT inhibition.

EIF1 Loop 2 interactions with Met-tRNAi control the accuracy of start codon selection by the scanning preinitiation complex.

The eukaryotic 43S preinitiation complex (PIC), bearing initiator methionyl transfer RNA (Met-tRNAi) in a ternary complex (TC) with eukaryotic initiation factor 2 (eIF2)-GTP, scans the mRNA leader for an AUG codon in favorable context. AUG recognition evokes rearrangement from an open PIC conformation with TC in a "POUT" state to a closed conformation with TC more tightly bound in a "PIN" state. eIF1 binds to the 40S subunit and exerts a dual role of enhancing TC binding to the open PIC conformation while antagonizing the PIN state, necessitating eIF1 dissociation for start codon selection. Structures of reconstituted PICs reveal juxtaposition of eIF1 Loop 2 with the Met-tRNAi D loop in the PIN state and predict a distortion of Loop 2 from its conformation in the open complex to avoid a clash with Met-tRNAi We show that Ala substitutions in Loop 2 increase initiation at both near-cognate UUG codons and AUG codons in poor context. Consistently, the D71A-M74A double substitution stabilizes TC binding to 48S PICs reconstituted with mRNA harboring a UUG start codon, without affecting eIF1 affinity for 40S subunits. Relatively stronger effects were conferred by arginine substitutions; and no Loop 2 substitutions perturbed the rate of TC loading on scanning 40S subunits in vivo. Thus, Loop 2-D loop interactions specifically impede Met-tRNAi accommodation in the PIN state without influencing the POUT mode of TC binding; and Arg substitutions convert the Loop 2-tRNAi clash to an electrostatic attraction that stabilizes PIN and enhances selection of poor start codons in vivo.

EIF1A residues implicated in cancer stabilize translation preinitiation complexes and favor suboptimal initiation sites in yeast.

The translation pre-initiation complex (PIC) scans the mRNA for an AUG codon in favorable context, and AUG recognition stabilizes a closed PIC conformation. The unstructured N-terminal tail (NTT) of yeast eIF1A deploys five basic residues to contact tRNAi, mRNA, or 18S rRNA exclusively in the closed state. Interestingly, EIF1AX mutations altering the human eIF1A NTT are associated with uveal melanoma (UM). We found that substituting all five basic residues, and seven UM-associated substitutions, in yeast eIF1A suppresses initiation at near-cognate UUG codons and AUGs in poor context. Ribosome profiling of NTT substitution R13P reveals heightened discrimination against unfavorable AUG context genome-wide. Both R13P and K16D substitutions destabilize the closed complex at UUG codons in reconstituted PICs. Thus, electrostatic interactions involving the eIF1A NTT stabilize the closed conformation and promote utilization of suboptimal start codons. We predict UM-associated mutations alter human gene expression by increasing discrimination against poor initiation sites.

IF2 and unique features of initiator tRNAfMet help establish the translational reading frame

ABSTRACTTranslation begins at AUG, GUG, or UUG codons in bacteria. Start codon recognition occurs in the P site, which may help explain this first-position degeneracy. However, the molecular basis of start codon specificity remains unclear. In this study, we measured the codon dependence of 30S•mRNA•tRNAfMet and 30S•mRNA•tRNAMet complex formation. We found that complex stability varies over a large range with initiator tRNAfMet, following the same trend as reported previously for initiation rate in vivo (AUG > GUG, UUG > CUG, AUC, AUA > ACG). With elongator tRNAMet, the codon dependence of binding differs qualitatively, with virtually no discrimination between GUG and CUG. A unique feature of initiator tRNAfMet is a series of three G-C basepairs in the anticodon stem, which are known to be important for efficient initiation in vivo. A mutation targeting the central of these G-C basepairs causes the mRNA binding specificity pattern to change in a way reminiscent of elongator tRNAMet. Unexpectedly, for certain complexes containing fMet-tRNAfMet, we observed mispositioning of mRNA, such that codon 2 is no longer programmed in the A site. This mRNA mispositioning is exacerbated by the anticodon stem mutation and suppressed by IF2. These findings suggest that both IF2 and the unique anticodon stem of fMet-tRNAfMet help constrain mRNA positioning to set the correct reading frame during initiation.

Idiosyncratic recognition of UUG/UUA codons by modified nucleoside 5-taurinomethyluridine, τm5U present at 'wobble' position in anticodon loop of tRNALeu: A molecular modeling approach.

Lack of naturally occurring modified nucleoside 5-taurinomethyluridine (τm5U) at the ‘wobble’ 34th position in tRNALeu causes mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes (MELAS). The τm5U34 specifically recognizes UUG and UUA codons. Structural consequences of τm5U34 to read cognate codons have not been studied so far in detail at the atomic level. Hence, 50ns multiple molecular dynamics (MD) simulations of various anticodon stem loop (ASL) models of tRNALeu in presence and absence of τm5U34 along with UUG and UUA codons were performed to explore the dynamic behaviour of τm5U34 during codon recognition process. The MD simulation results revealed that τm5U34 recognizes G/A ending codons by ‘wobble’ as well as a novel ‘single’ hydrogen bonding interactions. RMSD and RMSF values indicate the comparative stability of the ASL models containing τm5U34 modification over the other models, lacking τm5U34. Another MD simulation study of 55S mammalian mitochondrial rRNA with tRNALeu showed crucial interactions between the A-site residues, A918, A919, G256 and codon-anticodon bases. Thus, these results could improve our understanding about the decoding efficiency of human mt tRNALeu with τm5U34 to recognize UUG and UUA codons.

Impaired Energy Production Contributes to Development of Failure in Taurine Deficient Heart.

Taurine forms a conjugate in the mitochondria with a uridine residue in the wobble position of tRNALeu(UUR). The resulting product, 5-taurinomethyluridine tRNALeu(UUR), increases the interaction between the UUG codon and AAU anticodon of tRNALeu(UUR), thereby improving the decoding of the UUG codon. We have shown that the protein most affected by the taurine conjugation product is ND6, which is a subunit of complex I of the respiratory chain. Thus, taurine deficiency exhibits reduced respiratory chain function. Based on these findings, we proposed that the taurine deficient heart is energy deficient. To test this idea, hearts were perfused with buffer containing acetate and glucose as substrates. The utilization of both substrates, as well as the utilization of endogenous lipids, was significantly reduced in the taurine deficient heart. This led to a 25% decrease in ATP production, an effect primarily caused by diminished aerobic metabolism and respiratory function. In addition, inefficient oxidative phosphorylation causes a further decrease in ATP generation. The data support the idea that reductions in energy metabolism, including oxidative phosphorylation, ATP generation and high energy phosphate content, contribute to the severity of the cardiomyopathy. The findings are also consistent with the hypothesis that taurine deficiency and reduced myocardial energy content increases mortality of the taurine deficient, failing heart. The clinical implications of these findings are addressed.

Forced Ambiguity of the Leucine Codons for Multiple-Site-Specific Incorporation of a Noncanonical Amino Acid.

Multiple-site-specific incorporation of a noncanonical amino acid into a recombinant protein would be a very useful technique to generate multiple chemical handles for bioconjugation and multivalent binding sites for the enhanced interaction. Previously combination of a mutant yeast phenylalanyl-tRNA synthetase variant and the yeast phenylalanyl-tRNA containing the AAA anticodon was used to incorporate a noncanonical amino acid into multiple UUU phenylalanine (Phe) codons in a site-specific manner. However, due to the less selective codon recognition of the AAA anticodon, there was significant misincorporation of a noncanonical amino acid into unwanted UUC Phe codons. To enhance codon selectivity, we explored degenerate leucine (Leu) codons instead of Phe degenerate codons. Combined use of the mutant yeast phenylalanyl-tRNA containing the CAA anticodon and the yPheRS_naph variant allowed incorporation of a phenylalanine analog, 2-naphthylalanine, into murine dihydrofolate reductase in response to multiple UUG Leu codons, but not to other Leu codon sites. Despite the moderate UUG codon occupancy by 2-naphthylalaine, these results successfully demonstrated that the concept of forced ambiguity of the genetic code can be achieved for the Leu codons, available for multiple-site-specific incorporation.

Three members of polyamine modulon under oxidative stress conditions: two transcription factors (SoxR and EmrR) and a glutathione synthetic enzyme (GshA).

Members of polyamine modulon whose synthesis is enhanced at the level of translation were looked for under oxidative stress conditions caused by 0.6 μM K2TeO3. When an Escherichia coli polyamine-requiring mutant MA261 was cultured in the presence of K2TeO3, the degree of polyamine stimulation of cell growth was greater than in cells cultured in the absence of K2TeO3. Under these conditions, synthesis of SoxR, a transcriptional factor for expression of the superoxide response regulon, EmrR, a negative transcriptional factor for expression of the genes for drug excretion proteins, EmrA and EmrB, and of GshA, γ-glutamylcysteine synthetase necessary for glutathione (GSH) synthesis, were stimulated by polyamines at the level of translation. Polyamine stimulation of SoxR and EmrR synthesis was dependent on the existence of an unusually located Shine-Dalgarno (SD) sequence in soxR and emrR mRNAs. Polyamine stimulation of GshA synthesis was due to the existence of the inefficient initiation codon UUG instead of AUG. Polyamine stimulation of the synthesis of EmrR was mainly observed at the logarithmic phase of growth, while that of the synthesis of SoxR and GshA was at the stationary phase. These results strongly suggest that polyamines are involved in easing of oxidative stress through stimulation of synthesis of SoxR, EmrR and GshA together with RpoS, previously found as a member of polyamine modulon at the stationary phase.

Eukaryotic translation initiation factor eIF5 promotes the accuracy of start codon recognition by regulating Pi release and conformational transitions of the preinitiation complex.

eIF5 is the GTPase activating protein (GAP) for the eIF2·GTP·Met-tRNAiMet ternary complex with a critical role in initiation codon selection. Previous work suggested that the eIF5 mutation G31R/SUI5 elevates initiation at UUG codons by increasing GAP function. Subsequent work implicated eIF5 in rearrangement of the preinitiation complex (PIC) from an open, scanning conformation to a closed state at AUG codons, from which Pi is released from eIF2·GDP·Pi. To identify eIF5 functions crucial for accurate initiation, we investigated the consequences of G31R on GTP hydrolysis and Pi release, and the effects of intragenic G31R suppressors on these reactions, and on the partitioning of PICs between open and closed states. eIF5-G31R altered regulation of Pi release, accelerating it at UUG while decreasing it at AUG codons, consistent with its ability to stabilize the closed complex at UUG. Suppressor G62S mitigates both defects of G31R, accounting for its efficient suppression of UUG initiation in G31R,G62S cells; however suppressor M18V impairs GTP hydrolysis with little effect on PIC conformation. The strong defect in GTP hydrolysis conferred by M18V likely explains its broad suppression of Sui− mutations in numerous factors. We conclude that both of eIF5's functions, regulating Pi release and stabilizing the closed PIC conformation, contribute to stringent AUG selection in vivo.

Clinical significance of taurine

The biosynthesis of the b-amino acid, taurine, from cysteine is a two-step process, involving the oxidation of the sulfhydryl group and the decarboxylation of the amino acid (Stipanuk 2004). Although taurine was considered merely an end-product of cysteine degradation for many years, the detection of damaged retina in taurine deficient cats, convinced scientists that taurine was likely a physiologically important compound. This idea was reinforced over the next decade, as taurine was identified as an essential nutrient in several species, including the cat (Hayes and Carey 1975; Knopf et al. 1978; Sturman 1986), certain dogs (Gavaghan and Kittleson 1997; Backus et al. 2003; Belanger et al. 2005), the fox (Moise et al. 1991), some monkeys (Hayes et al. 1980; Stephan et al. 1981; Imaki et al. 1987) and more recently the anteater (Nofs et al. 2013). Among the defects associated with taurine deficiency have been retinal and tapetum degeneration (Hayes and Carey 1975; Sturman 1986), cardiac dysfunction (Pion et al. 1987, 1992; Novotny et al. 1991), immune deficiency (Schuller-Levis et al. 1990), muscle atrophy (Ito et al. 2008), premature aging (Ito et al. 2013a) and impaired reproduction (Hayes et al. 1980; Sturman 1986). The identification of taurine as an essential nutrient in some species and a semi-essential nutrient in man prompted the search for new physiological functions of the amino acid. Several of those newly discovered functions have been tied to taurine conjugation. One reaction involves the detoxification of hypochlorous acid by taurine resulting in the formation of taurine chloramine, a product that has the added benefit of possessing anti-inflammatory activity (Schuller-Levis and Park 2003; Marcinkiewicz and Kontny 2013; Kim and Cha 2014). Taurine also forms a conjugate (5-taurinomethyluridine) with the wobble position uridine of tRNA, a reaction that enhances the interaction between the modified uridine and guanine (Kirino et al. 2005; Kurata et al. 2008; Schaffer et al. 2013). Biochemically, this enhances the binding of the UUG codon to the taurine-modified AAU anticodon of tRNA, facilitating UUG decoding. When the formation of 5-taurinomethyluridine-tRNA is abolished, the biosynthesis of certain mitochondria encoded proteins dramatically declines, respiratory function falls, ATP generation decreases and the generation of oxidants by the respiratory chain increases (Jong et al. 2012; Schaffer et al. 2013). Another important function of taurine is the detoxification of xenobiotics and the neutralization of toxic aldehydes (Miyazaki and Matsuzaki 2013). Because taurine is found at a very high concentration within most cells (mM range) it is not surprising that it also serves as a key organic osmolyte (Lang et al. 1998; Huang et al. 2006). In the central nervous system, taurine specifically functions as a neuromodulator, interacting and altering the actions of the GABAA receptor and the glycine receptor (Menzie et al. 2013). The recognition that taurine is an essential nutrient in cats and certain monkeys initially raised questions about the nutritional status of taurine in humans, who generally consume large amounts of taurine in their diet but possess a limited capacity to synthesize taurine. However, unlike taurine-dependent species, such as the cat, humans lose S. W. Schaffer (&) Department of Pharmacology, School of Medicine, University of South Alabama, Mobile, AL 36695, USA e-mail: sschaffe@southalabama.edu

β-Hairpin Loop of Eukaryotic Initiation Factor 1 (eIF1) Mediates 40 S Ribosome Binding to Regulate Initiator tRNAMet Recruitment and Accuracy of AUG Selection in Vivo

Recognition of the translation initiation codon is thought to require dissociation of eIF1 from the 40 S ribosomal subunit, enabling irreversible GTP hydrolysis (Pi release) by the eIF2·GTP·Met-tRNAi ternary complex (TC), rearrangement of the 40 S subunit to a closed conformation incompatible with scanning, and stable binding of Met-tRNAi to the P site. The crystal structure of a Tetrahymena 40 S·eIF1 complex revealed several basic amino acids in eIF1 contacting 18 S rRNA, and we tested the prediction that their counterparts in yeast eIF1 are required to prevent premature eIF1 dissociation from scanning ribosomes at non-AUG triplets. Supporting this idea, substituting Lys-60 in helix α1, or either Lys-37 or Arg-33 in β-hairpin loop-1, impairs binding of yeast eIF1 to 40 S·eIF1A complexes in vitro, and it confers increased initiation at UUG codons (Sui(-) phenotype) or lethality, in a manner suppressed by overexpressing the mutant proteins or by an eIF1A mutation (17-21) known to impede eIF1 dissociation in vitro. The eIF1 Sui(-) mutations also derepress translation of GCN4 mRNA, indicating impaired ternary complex loading, and this Gcd(-) phenotype is likewise suppressed by eIF1 overexpression or the 17-21 mutation. These findings indicate that direct contacts of eIF1 with 18 S rRNA seen in the Tetrahymena 40 S·eIF1 complex are crucial in yeast to stabilize the open conformation of the 40 S subunit and are required for rapid TC loading and ribosomal scanning and to impede rearrangement to the closed complex at non-AUG codons. Finally, we implicate the unstructured N-terminal tail of eIF1 in blocking rearrangement to the closed conformation in the scanning preinitiation complex.