Portrait of Dr Tobias von der Haar

Dr Tobias von der Haar

Reader in Systems Biology
Director of Research

About

Dr Tobias von der Haar received a Diploma in Biology from the University of Bielefeld (Germany) in 1994. He received his PhD on the regulation of protein synthesis in baker’s yeast from UMIST (Manchester, UK) in 1998. Tobias established his own lab in the School of Biosciences at the University of Kent in 2005 under a Wellcome Trust Career Development Fellowship. He is currently Reader in Systems Biology, a member of the Kent Fungal Group and of the Industrial Biotechnology research group, and serves as Director of Research for the School of Biosciences.

ORCID ID: 0000-0002-6031-9254

Research interests

His research interests include the mechanism of protein synthesis in eukaryotic cells, and how protein synthesis can be manipulated for the production of useful proteins.
Ribosomes and tRNAs are the central molecules required for protein synthesis to work. Ribosomes have the interesting feature that they are both catalysts (they make peptide bonds in the new protein) and molecular motors (they move along the mRNA in order to read the genetic code). One of the central areas of interest in my lab is how the movement along the mRNA and the catalysis of peptide bond formation interact. Our main tools to study this are computational models which enable us to predict ribosome movement, reporter assays which allow us to test predictions from the computational models, and genome-wide omics approaches which enable us to ask questions about the regulation of individual genes in great detail.
In addition to ribosome activity, we also study how proteins are activated in a number of post-translational processes including disulfide bond formation and the introduction of iron sulfur clusters. For all these processes, we want to understand the basic biology of organisms, but also to exploit this understanding for generating useful applications. Examples of such applications include the making of protein-based drugs, the reprogramming of metabolism for synthetic biology, and the degradation of problematic materials like plastics.

Supervision

MSc-R projects available for 2019/20

Towards the biological degradation of plastic materials Plastic materials are a ubiquitous environmental problem, in part due to their strong resistance to biological degradation. However, organisms that can degrade plastics to a small extent are known. This project will aim to improve the natural plastic degrading activities by applying biological engineering strategies in a number of microbial host species.
Additional research costs: £1200
tRNA biology, an understudied hub of gene expression control Transfer RNAs (tRNAs) have an essential role in delivering amino acids to the ribosome during protein synthesis. Due to their complicated biology they have long been poorly understood, but recent technical advances in deep sequencing and mass spectrometric approaches have revealed them to be central biological regulators with fascinating properties. This project will investigate how the base composition of tRNA changes in response to specific stresses, and how this in turn regulates the expression of specific target genes. Additional research costs: £1200
Genes, health, disease and ageing: a data science approach Current biological research is generating relentless data streams, which could yield deep insights into our biology - unfortunately, such insights are usually hidden deep within the complex data structures. This has opened the field of biological data science, which uses computational and related methods to derive insights from biological data. This project will use publicly available data to investigate how protein synthesis is altered in the contexts of healthy development, disease, and ageing. Additional research costs: £1200

Professional

Tobias has a strong interest in the development of practices and policies relating to postgraduate study. He recently served as Director of Graduate studies for the School of Biosciences (2013-2016), is external examiner for postgraduate degrees at other universities, and is currently a member of the Bioscience Skills and Careers advisory panel of the BBSRC. 

Publications

Showing 50 of 52 total publications in the Kent Academic Repository. View all publications.

Article

  • Xie, J. et al. (2019). Regulation of the Elongation Phase of Protein Synthesis Enhances Translation Accuracy and Modulates Lifespan. Current Biology [Online] 29:1-13. Available at: http://dx.doi.org/10.1016/j.cub.2019.01.029.
    Maintaining accuracy during protein synthesis is crucial to avoid producing misfolded and/or non-functional proteins. The target of rapamycin complex 1 (TORC1) pathway and the activity of the protein synthesis machinery are known to negatively regulate lifespan in many organisms, although the precise mechanisms involved remain unclear. Mammalian TORC1 signaling accelerates the elongation stage of protein synthesis by inactivating eukaryotic elongation factor 2 kinase (eEF2K), which, when active, phosphorylates and inhibits eEF2, which mediates the movement of ribosomes along mRNAs, thereby slowing down the rate of elongation. We show that eEF2K enhances the accuracy of protein synthesis under a range of conditions and in several cell types. For example, our data reveal it links mammalian (m)TORC1 signaling to the accuracy of translation. Activation of eEF2K decreases misreading or termination readthrough errors during elongation, whereas knocking down or knocking out eEF2K increases their frequency. eEF2K also promotes the correct recognition of start codons in mRNAs. Reduced translational fidelity is known to correlate with shorter lifespan. Consistent with this, deletion of the eEF2K ortholog or other factors implicated in translation fidelity in Caenorhabditis elegans decreases lifespan, and eEF2K is required for lifespan extension induced by nutrient restriction. Our data uncover a novel mechanism linking nutrient supply, mTORC1 signaling, and the elongation stage of protein synthesis, which enhances the accuracy of protein synthesis. Our data also indicate that modulating translation elongation and its fidelity affects lifespan.
  • Jossé, L., Singh, T. and von der Haar, T. (2019). Experimental determination of codon usage?dependent selective pressure on high copy?number genes in Saccharomyces cerevisiae. Yeast [Online] 36:43-51. Available at: https://doi.org/10.1002/yea.3373.
    One of the central hypotheses in the theory of codon usage evolution is that in highly expressed genes particular codon usage patterns arise because they facilitate efficient gene expression and are thus selected for in evolution. Here we use plasmid copy number assays and growth rate measurements to explore details of the relationship between codon usage, gene expression level, and selective pressure in Saccharomyces cerevisiae. We find that when high expression levels are required optimal codon usage is beneficial and provides a fitness advantage, consistent with evolutionary theory. However, when high expression levels are not required, optimal codon usage is surprisingly and strongly selected against. We show that this selection acts at the level of protein synthesis, and we exclude a number of molecular mechanisms as the source for this negative selective pressure including nutrient and ribosome limitations and proteotoxicity effects. These findings inform our understanding of the evolution of codon usage bias, as well as the design of recombinant protein expression systems.
  • Khan, M., Spurgeon, S. and von der Haar, T. (2018). Origins of robustness in translational control via eukaryotic translation initiation factor (eIF) 2. Journal of Theoretical Biology [Online] 445:92-102. Available at: https://doi.org/10.1016/j.jtbi.2018.02.020.
    Phosphorylation of eukaryotic translation initiation factor 2 (eIF2) is one of the best studied and most widely used means for regulating protein synthesis activity in eukaryotic cells. This pathway regulates protein synthesis in response to stresses, viral infections, and nutrient depletion, among others. We present analyses of an ordinary differential equation-based model of this pathway, which aim to identify its principal robustness-conferring features. Our analyses indicate that robustness is a distributed property, rather than arising from the properties of any one individual pathway species. However, robustness-conferring properties are unevenly distributed between the different species, and we identify a guanine nucleotide dissociation inhibitor (GDI) complex as a species that likely contributes strongly to the robustness of the pathway. Our analyses make further predictions on the dynamic response to different types of kinases that impinge on eIF2.
  • von der Haar, T. et al. (2017). The control of translational accuracy is a determinant of healthy ageing in yeast. Open Biology [Online] 7:160291. Available at: http://dx.doi.org/10.1098/rsob.160291.
    Life requires the maintenance of molecular function in the face of stochastic processes that tend to adversely affect macromolecular integrity. This is particularly relevant during ageing, as many cellular functions decline with age, including growth, mitochondrial function and energy metabolism. Protein synthesis must deliver functional proteins at all times, implying that the effects of protein synthesis errors like amino acid misincorporation and stop-codon read-through must be minimized during ageing. Here we show that loss of translational accuracy accelerates the loss of viability in stationary phase yeast. Since reduced translational accuracy also reduces the folding competence of at least some proteins, we hypothesize that negative interactions between translational errors and age-related protein damage together overwhelm the cellular chaperone network. We further show that multiple cellular signalling networks control basal error rates in yeast cells, including a ROS signal controlled by mitochondrial activity, and the Ras pathway. Together, our findings indicate that signalling pathways regulating growth, protein homeostasis and energy metabolism may jointly safeguard accurate protein synthesis during healthy ageing.
  • Saintas, E. et al. (2017). Acquired resistance to oxaliplatin is not directly associated with increased resistance to DNA damage in SK-N-ASrOXALI4000, a newly established oxaliplatin-resistant sub-line of the neuroblastoma cell line SK-N-AS. PloS one [Online] 12:e0172140. Available at: http://dx.doi.org/10.1371/journal.pone.0172140.
    The formation of acquired drug resistance is a major reason for the failure of anti-cancer therapies after initial response. Here, we introduce a novel model of acquired oxaliplatin resistance, a sub-line of the non-MYCN-amplified neuroblastoma cell line SK-N-AS that was adapted to growth in the presence of 4000 ng/mL oxaliplatin (SK-N-ASrOXALI4000). SK-N-ASrOXALI4000 cells displayed enhanced chromosomal aberrations compared to SK-N-AS, as indicated by 24-chromosome fluorescence in situ hybridisation. Moreover, SK-N-ASrOXALI4000 cells were resistant not only to oxaliplatin but also to the two other commonly used anti-cancer platinum agents cisplatin and carboplatin. SK-N-ASrOXALI4000 cells exhibited a stable resistance phenotype that was not affected by culturing the cells for 10 weeks in the absence of oxaliplatin. Interestingly, SK-N-ASrOXALI4000 cells showed no cross resistance to gemcitabine and increased sensitivity to doxorubicin and UVC radiation, alternative treatments that like platinum drugs target DNA integrity. Notably, UVC-induced DNA damage is thought to be predominantly repaired by nucleotide excision repair and nucleotide excision repair has been described as the main oxaliplatin-induced DNA damage repair system. SK-N-ASrOXALI4000 cells were also more sensitive to lysis by influenza A virus, a candidate for oncolytic therapy, than SK-N-AS cells. In conclusion, we introduce a novel oxaliplatin resistance model. The oxaliplatin resistance mechanisms in SK-N-ASrOXALI4000 cells appear to be complex and not to directly depend on enhanced DNA repair capacity. Models of oxaliplatin resistance are of particular relevance since research on platinum drugs has so far predominantly focused on cisplatin and carboplatin.
  • Saintas, E. et al. (2017). Acquired resistance to oxaliplatin is not directly associated with increased resistance to DNA damage in SK-N-ASrOXALI4000, a newly established oxaliplatin-resistant sub-line of the neuroblastoma cell line SK-N-AS. PLoS ONE [Online] 12:e0172140. Available at: http://dx.doi.org/10.1371/journal.pone.0172140.
    The formation of acquired drug resistance is a major reason for the failure of anti-cancer therapies after initial response. Here, we introduce a novel model of acquired oxaliplatin resistance, a sub-line of the non-MYCN-amplified neuroblastoma cell line SK-N-AS that was adapted to growth in the presence of 4000 ng/mL oxaliplatin (SK-N-ASrOXALI4000). SK-N-ASrOXALI4000 cells displayed enhanced chromosomal aberrations compared to SK-N-AS, as indicated by 24-chromosome fluorescence in situ hybridisation. Moreover, SK-N-ASrOXALI4000 cells were resistant not only to oxaliplatin but also to the two other commonly used anti-cancer platinum agents cisplatin and carboplatin. SK-N-ASrOXALI4000 cells exhibited a stable resistance phenotype that was not affected by culturing the cells for 10 weeks in the absence of oxaliplatin. Interestingly, SK-N-ASrOXALI4000 cells showed no cross resistance to gemcitabine and increased sensitivity to doxorubicin and UVC radiation, alternative treatments that like platinum drugs target DNA integrity. Notably, UVC-induced DNA damage is thought to be predominantly repaired by nucleotide excision repair and nucleotide excision repair has been described as the main oxaliplatin-induced DNA damage repair system. SK-N-ASrOXALI4000 cells were also more sensitive to lysis by influenza A virus, a candidate for oncolytic therapy, than SK-N-AS cells. In conclusion, we introduce a novel oxaliplatin resistance model. The oxaliplatin resistance mechanisms in SK-N-ASrOXALI4000 cells appear to be complex and not to directly depend on enhanced DNA repair capacity. Models of oxaliplatin resistance are of particular relevance since research on platinum drugs has so far predominantly focused on cisplatin and carboplatin.
  • Bastide, A. et al. (2017). RTN3 Is a Novel Cold-Induced Protein and Mediates Neuroprotective Effects of RBM3. Current Biology [Online] 27:638-650. Available at: https://doi.org/10.1016/j.cub.2017.01.047.
    Cooling and hypothermia are profoundly neuroprotective, mediated, at least in part, by the cold shock protein, RBM3. However, the neuroprotective effector proteins induced by RBM3 and the mechanisms by which mRNAs encoding cold shock proteins escape cooling-induced translational repression are unknown. Here, we show that cooling induces reprogramming of the translatome, including the upregulation of a new cold shock protein, RTN3, a reticulon protein implicated in synapse formation. We report that this has two mechanistic components. Thus, RTN3 both evades cooling-induced translational elongation repression and is also bound by RBM3, which drives the increased expression of RTN3. In mice, knockdown of RTN3 expression eliminated cooling-induced neuroprotection. However, lentivirally mediated RTN3 overexpression prevented synaptic loss and cognitive deficits in a mouse model of neurodegeneration, downstream and independently of RBM3. We conclude that RTN3 expression is a mediator of RBM3-induced neuroprotection, controlled by novel mechanisms of escape from translational inhibition on cooling.
  • Tarrant, D. et al. (2016). Inappropriate expression of the translation elongation factor 1A disrupts genome stability and metabolism. Journal of Cell Science [Online] 129:4455-4465. Available at: http://doi.org/10.1242/jcs.192831.
    The translation elongation factor eEF1A is one of the most abundant proteins found within cells and its role within protein synthesis is well documented. Levels of eEF1A are tightly controlled, with inappropriate expression linked to oncogenesis. However the mechanisms by which increased eEF1A expression alter cell behaviour are unknown. Our analyses in yeast suggest that elevation of eEF1A levels lead to stabilisation of the spindle pole body and changes in nuclear organisation. Elevation of eEF1A2 also leads to altered nuclear morphology in cultured human cells suggesting a conserved role in maintaining genome stability. Gene expression and metabolomic analyses reveal that the level of eEF1A is crucial for the maintenance of metabolism and amino acid levels in yeast, most likely via its role in the control of vacuole function. Increased eEF1A2 levels trigger lysosome biogenesis in cultured human cells, also suggesting a conserved role within metabolic control mechanisms. Together our data suggest that the control of eEF1A levels is important for the maintenance of a number of cell functions out-with translation, whose de-regulation may contribute to its oncogenic properties.
  • Bill, R. and von der Haar, T. (2015). Hijacked then lost in translation: the plight of the recombinant host cell in membrane protein structural biology projects. Current Opinion in Structural Biology [Online] 32:147-155. Available at: http://doi.org/10.1016/j.sbi.2015.04.003.
    Membrane protein structural biology is critically dependent upon the supply of high-quality protein. Over the last few years, the value of crystallising biochemically characterised, recombinant targets that incorporate stabilising mutations has been established. Nonetheless, obtaining sufficient yields of many recombinant membrane proteins is still a major challenge. Solutions are now emerging based on an improved understanding of recombinant host cells; as a ‘cell factory’ each cell is tasked with managing limited resources to simultaneously balance its own growth demands with those imposed by an expression plasmid. This review examines emerging insights into the role of translation and protein folding in defining high-yielding recombinant membrane protein production in a range of host cells.
  • Beznoskova, P. et al. (2015). Translation initiation factor eIF3 promotes programmed stop codon readthrough. Nucleic Acids Research [Online] 43:5099-5111. Available at: http://nar.oxfordjournals.org/content/43/10/5099.
    Programmed stop codon readthrough is a post-transcription regulatory mechanism specifically increasing proteome diversity by creating a pool of C-terminally extended proteins. During this process, the stop codon is decoded as a sense codon by a near-cognate tRNA, which programs the ribosome to continue elongation. The efficiency of competition for the stop codon between release factors (eRFs) and near-cognate tRNAs is largely dependent on its nucleotide context; however, the molecular mechanism underlying this process is unknown. Here, we show that it is the translation initiation (not termination) factor, namely eIF3, which critically promotes programmed readthrough on all three stop codons. In order to do so, eIF3 must associate with pre-termination complexes where it interferes with the eRF1 decoding of the third/wobble position of the stop codon set in the unfavorable termination context, thus allowing incorporation of near-cognate tRNAs with a mismatch at the same position. We clearly demonstrate that efficient readthrough is enabled by near-cognate tRNAs with a mismatch only at the third/wobble position. Importantly, the eIF3 role in programmed readthrough is conserved between yeast and humans.
  • Blanchet, S. et al. (2015). New insights into stop codon recognition by eRF1. Nucleic Acids Research [Online] 43:3298-3308. Available at: http://dx.doi.org/10.1093/nar/gkv154.
    In eukaryotes, translation termination is performed by eRF1, which recognizes stop codons via its N-terminal domain. Many previous studies based on point mutagenesis, cross-linking experiments or eRF1 chimeras have investigated the mechanism by which the stop signal is decoded by eRF1. Conserved motifs, such as GTS and YxCxxxF, were found to be important for termination efficiency, but the recognition mechanism remains unclear. We characterized a region of the eRF1 N-terminal domain, the P1 pocket, that we had previously shown to be involved in termination efficiency. We performed alanine scanning mutagenesis of this region, and we quantified in vivo readthrough efficiency for each alanine mutant. We identified two residues, arginine 65 and lysine 109, as critical for recognition of the three stop codons. We also demonstrated a role for the serine 33 and serine 70 residues in UGA decoding in vivo. NMR analysis of the alanine mutants revealed that the correct conformation of this region was controlled by the YxCxxxF motif. By combining our genetic data with a structural analysis of eRF1 mutants, we were able to formulate a new model in which the stop codon interacts with eRF1 through the P1 pocket.
  • Mead, E. et al. (2014). Control and regulation of mRNA translation. Biochemical Society transactions [Online] 42:151-154. Available at: http://dx.doi.org/10.1042/BST20130259.
    Translational control is central to the gene expression pathway and was the focus of the 2013 annual Translation UK meeting held at the University of Kent. The meeting brought together scientists at all career stages to present and discuss research in the mRNA translation field, with an emphasis on the presentations on the research of early career scientists. The diverse nature of this field was represented by the broad range of papers presented at the meeting. The complexity of mRNA translation and its control is emphasized by the interdisciplinary research approaches required to address this area with speakers highlighting emerging systems biology techniques and their application to understanding mRNA translation and the network of pathways controlling it.
  • Tarrant, D. and von der Haar, T. (2014). Synonymous codons, ribosome speed, and eukaryotic gene expression regulation. Cellular and Molecular Life Sciences [Online] 71:4195-4206. Available at: http://dx.doi.org/10.1007/s00018-014-1684-2.
    Quantitative control of gene expression occurs at multiple levels, including the level of translation. Within the overall process of translation, most identified regulatory processes impinge on the initiation phase. However, recent studies have revealed that the elongation phase can also regulate translation if elongation and initiation occur with specific, not mutually compatible rate parameters. Translation elongation then limits the overall amount of protein that can be made from an mRNA. Several recently discovered control mechanisms of biological pathways are based on such elongation control. Here, we review the molecular mechanisms that determine ribosome speed in eukaryotic organisms, and discuss under which conditions ribosome speed can become the controlling parameter of gene expression levels.
  • Chu, D., Thompson, J. and von der Haar, T. (2014). Charting the dynamics of translation. Bio Systems [Online] 119:1-9. Available at: http://dx.doi.org/10.1016/j.biosystems.2014.02.005.
    Codon usage bias (CUB) is the well-known phenomenon that the frequency of synonymous codons is unequal. This is presumably the result of adaptive pressures favouring some codons over others. The underlying reason for this pressure is unknown, although a large number of possible driver mechanisms have been proposed. According to one hypothesis, the decoding time could be such a driver. A tacit assumption of this hypothesis is that faster codons lead to a higher translation rate which in turn is more resource efficient. While it is generally assumed that there is such a link, there are no rigorous studies to establish under which conditions the link between translation speed and rate actually exists. Using a computational simulation model and explicitly calculated codon decoding times, this contribution maps the entire range of dynamical regimes of translation. These simulations make it possible to understand precisely under which conditions translation speed and rate are linked.
  • Chu, D. et al. (2014). Translation elongation can control translation initiation on eukaryotic mRNAs. EMBO Journal [Online] 33:21-34. Available at: http://dx.doi.org/10.1002/embj.201385651.
    Synonymous codons encode the same amino acid, but differ in other biophysical properties. The evolutionary selection of codons whose properties are optimal for a cell generates the phenomenon of codon bias. Although recent studies have shown strong effects of codon usage changes on protein expression levels and cellular physiology, no translational control mechanism is known that links codon usage to protein expression levels. Here, we demonstrate a novel translational control mechanism that responds to the speed of ribosome movement immediately after the start codon. High initiation rates are only possible if start codons are liberated sufficiently fast, thus accounting for the observation that fast codons are overrepresented in highly expressed proteins. In contrast, slow codons lead to slow liberation of the start codon by initiating ribosomes, thereby interfering with efficient translation initiation. Codon usage thus evolved as a means to optimise translation on individual mRNAs, as well as global optimisation of ribosome availability.
  • von der Haar, T. and Kazana, E. (2014). The translational machinery is an optimized molecular network that affects cellular homoeostasis and disease. Biochemical Society Transactions [Online] 42:173-176. Available at: http://dx.doi.org/10.1042/BST20130131.
    Translation involves interactions between mRNAs, ribosomes, tRNAs and a host of translation factors. Emerging evidence on the eukaryotic translational machinery indicates that these factors are organized in a highly optimized network, in which the levels of the different factors are finely matched to each other. This optimal factor network is essential for producing proteomes that result in optimal fitness, and perturbations to the optimal network that significantly affect translational activity therefore result in non-optimal proteomes, fitness losses and disease. On the other hand, experimental evidence indicates that translation and cell growth are relatively robust to perturbations, and viability can be maintained even upon significant damage to individual translation factors. How the eukaryotic translational machinery is optimized, and how it can maintain optimization in the face of changing internal parameters, are open questions relevant to the interaction between translation and cellular disease states.
  • Beznoskova, P. et al. (2013). Translation Initiation Factors eIF3 and HCR1 Control Translation Termination and Stop Codon Read-Through in Yeast Cells. PLoS Genetics [Online] 9:e1003962. Available at: http://dx.doi.org/10.1371/journal.pgen.1003962.
    Translation is divided into initiation, elongation, termination and ribosome recycling. Earlier work implicated several eukaryotic initiation factors (eIFs) in ribosomal recycling in vitro. Here, we uncover roles for HCR1 and eIF3 in translation termination in vivo. A substantial proportion of eIF3, HCR1 and eukaryotic release factor 3 (eRF3) but not eIF5 (a well-defined “initiation-specific” binding partner of eIF3) specifically co-sediments with 80S couples isolated from RNase-treated heavy polysomes in an eRF1-dependent manner, indicating the presence of eIF3 and HCR1 on terminating ribosomes. eIF3 and HCR1 also occur in ribosome- and RNA-free complexes with both eRFs and the recycling factor ABCE1/RLI1. Several eIF3 mutations reduce rates of stop codon read-through and genetically interact with mutant eRFs. In contrast, a slow growing deletion of hcr1 increases read-through and accumulates eRF3 in heavy polysomes in a manner suppressible by overexpressed ABCE1/RLI1. Based on these and other findings we propose that upon stop codon recognition, HCR1 promotes eRF3·GDP ejection from the post-termination complexes to allow binding of its interacting partner ABCE1/RLI1. Furthermore, the fact that high dosage of ABCE1/RLI1 fully suppresses the slow growth phenotype of hcr1? as well as its termination but not initiation defects implies that the termination function of HCR1 is more critical for optimal proliferation than its function in translation initiation. Based on these and other observations we suggest that the assignment of HCR1 as a bona fide eIF3 subunit should be reconsidered. Together our work characterizes novel roles of eIF3 and HCR1 in stop codon recognition, defining a communication bridge between the initiation and termination/recycling phases of translation.
  • Hopker, J. et al. (2013). The influence of training status, age, and muscle fiber type on cycling efficiency and endurance performance. Journal of Applied Physiology [Online] 115:723-729. Available at: http://dx.doi.org/10.?1152/?japplphysiol.?00361.?2013.
    The purpose of this study was to assess the influence of age, training status, and muscle fiber-type distribution on cycling efficiency. Forty men were recruited into one of four groups: young and old trained cyclists, and young and old untrained individuals. All participants completed an incremental ramp test to measure their peak O2 uptake, maximal heart rate, and maximal minute power output; a submaximal test of cycling gross efficiency (GE) at a series of absolute and relative work rates; and, in trained participants only, a 1-h cycling time trial. Finally, all participants underwent a muscle biopsy of their right vastus lateralis muscle. At relative work rates, a general linear model found significant main effects of age and training status on GE (P < 0.01). The percentage of type I muscle fibers was higher in the trained groups (P < 0.01), with no difference between age groups. There was no relationship between fiber type and cycling efficiency at any work rate or cadence combination. Stepwise multiple regression indicated that muscle fiber type did not influence cycling performance (P > 0.05). Power output in the 1-h performance trial was predicted by average O2 uptake and GE, with standardized ?-coefficients of 0.94 and 0.34, respectively, although some mathematical coupling is evident. These data demonstrate that muscle fiber type does not affect cycling efficiency and was not influenced by the aging process. Cycling efficiency and the percentage of type I muscle fibers were influenced by training status, but only GE at 120 revolutions/min was seen to predict cycling performance.
  • Preiss, T. et al. (2013). Specialized Yeast Ribosomes: A Customized Tool for Selective mRNA Translation. PLoS ONE [Online] 8:e67609. Available at: http://dx.doi.org/10.1371/journal.pone.0067609.
    Evidence is now accumulating that sub-populations of ribosomes - so-called specialized ribosomes - can favour the translation of subsets of mRNAs. Here we use a large collection of diploid yeast strains, each deficient in one or other copy of the set of ribosomal protein (RP) genes, to generate eukaryotic cells carrying distinct populations of altered ‘specialized’ ribosomes. We show by comparative protein synthesis assays that different heterologous mRNA reporters based on luciferase are preferentially translated by distinct populations of specialized ribosomes. These mRNAs include reporters carrying premature termination codons (PTC) thus allowing us to identify specialized ribosomes that alter the efficiency of translation termination leading to enhanced synthesis of the wild-type protein. This finding suggests that these strains can be used to identify novel therapeutic targets in the ribosome. To explore this further we examined the translation of the mRNA encoding the extracellular matrix protein laminin ?3 (LAMB3) since a LAMB3-PTC mutant is implicated in the blistering skin disease Epidermolysis bullosa (EB). This screen identified specialized ribosomes with reduced levels of RP L35B as showing enhanced synthesis of full-length LAMB3 in cells expressing the LAMB3-PTC mutant. Importantly, the RP L35B sub-population of specialized ribosomes leave both translation of a reporter luciferase carrying a different PTC and bulk mRNA translation largely unaltered.
  • Chu, D. and von der Haar, T. (2012). The architecture of eukaryotic translation. Nucleic Acids Research [Online] 40:10098-10106. Available at: http://dx.doi.org/10.1093/nar/gks825.
    Translation in baker’s yeast involves the coordinated interaction of 200 000 ribosomes, 3 000 000 tRNAs and between 15 000 and 60 000 mRNAs. It is currently unknown whether this specific constellation of components has particular relevance for the requirements of the yeast proteome, or whether this is simply a frozen accident. Our study uses a computational simulation model of the genome-wide translational apparatus of yeast to explore quantitatively which combinations of mRNAs, ribosomes and tRNAs can produce viable proteomes. Surprisingly, we find that if we only consider total translational activity over time without regard to composition of the proteome, then there are many and widely differing combinations that can generate equivalent synthesis yields. In contrast, translational activity required for generating specific proteomes can only be achieved within a much more constrained parameter space. Furthermore, we find that strongly ribosome limited regimes are optimal for cells in that they are resource efficient and simplify the dynamics of the system.
  • Jossé, L. et al. (2012). Probing the role of structural features of mouse PrP in yeast by expression as Sup35-PrP fusions. Prion [Online] 6:201-210. Available at: http://dx.doi.org/10.4161/pri.19214.
    The yeast Saccharomyces cerevisiae is a tractable model organism in which both to explore the molecular mechanisms underlying the generation of disease-associated protein misfolding and to map the cellular responses to potentially toxic misfolded proteins. Specific targets have included proteins which in certain disease states form amyloids and lead to neurodegeneration. Such studies are greatly facilitated by the extensive ‘toolbox’ available to the yeast researcher that provides a range of cell engineering options. Consequently, a number of assays at the cell and molecular level have been set up to report on specific protein misfolding events associated with endogenous or heterologous proteins. One major target is the mammalian prion protein PrP because we know little about what specific sequence and/or structural feature(s) of PrP are important for its conversion to the infectious prion form, PrPSc. Here, using a study of the expression in yeast of fusion proteins comprising the yeast prion protein Sup35 fused to various regions of mouse PrP protein, we show how PrP sequences can direct the formation of non-transmissible amyloids and focus in particular on the role of the mouse octarepeat region. Through this study we illustrate the benefits and limitations of yeast-based models for protein misfolding disorders.
  • von der Haar, T. (2012). Mathematical and Computational Modelling of Ribosomal Movement and Protein Synthesis: an overview. Computational and Structural Biotechnology Journal [Online] 1:e201204002. Available at: http://dx.doi.org/10.5936/csbj.201204002.
    Translation or protein synthesis consists of a complex system of chemical reactions, which ultimately result in decoding of the mRNA and the production of a protein. The complexity of this reaction system makes it difficult to quantitatively connect its input parameters (such as translation factor or ribosome concentrations, codon composition of the mRNA, or energy availability) to output parameters (such as protein synthesis rates or ribosome densities on mRNAs). Mathematical and computational models of translation have now been used for nearly five decades to investigate translation, and to shed light on the relationship between the different reactions in the system. This review gives an overview over the principal approaches used in the modelling efforts, and summarises some of the major findings that were made.
  • Chu, D., Zabet, N. and von der Haar, T. (2012). A novel and versatile computational tool to model translation. Bioinformatics [Online] 28:292-293. Available at: http://dx.doi.org/10.1093/bioinformatics/btr650.
    Motivation: Much is now known about the mechanistic details of gene translation. There are also rapid advances in high-throughput technologies to determine quantitative aspects of the system. As a consequence-realistic and system-wide simulation models of translation are now feasible. Such models are also needed as devices to integrate a large volume of highly fragmented data known about translation.

    Software: In this application note, we present a novel, highly efficient software tool to model translation. The tool represents the main aspects of translation. Features include a representation of exhaustible tRNA pools, ribosome–ribosome interactions and differential initiation rates for different mRNA species. The tool is written in Java, and is hence portable and can be parameterized for any organism.

    Availability: The model can be obtained from the authors or directly downloaded from the authors' home-page (http://goo.gl/JUWvI).
  • Mead, E. et al. (2012). Experimental and in silico modelling analyses of the gene expression pathway for recombinant antibody and by-product production in NS0 cell lines. PloS one 7:e47422.
    Monoclonal antibodies are commercially important, high value biotherapeutic drugs used in the treatment of a variety of diseases. These complex molecules consist of two heavy chain and two light chain polypeptides covalently linked by disulphide bonds. They are usually expressed as recombinant proteins from cultured mammalian cells, which are capable of correctly modifying, folding and assembling the polypeptide chains into the native quaternary structure. Such recombinant cell lines often vary in the amounts of product produced and in the heterogeneity of the secreted products. The biological mechanisms of this variation are not fully defined. Here we have utilised experimental and modelling strategies to characterise and define the biology underpinning product heterogeneity in cell lines exhibiting varying antibody expression levels, and then experimentally validated these models. In undertaking these studies we applied and validated biochemical (rate-constant based) and engineering (nonlinear) models of antibody expression to experimental data from four NS0 cell lines with different IgG4 secretion rates. The models predict that export of the full antibody and its fragments are intrinsically linked, and cannot therefore be manipulated individually at the level of the secretory machinery. Instead, the models highlight strategies for the manipulation at the precursor species level to increase recombinant protein yields in both high and low producing cell lines. The models also highlight cell line specific limitations in the antibody expression pathway.
  • Mead, E. et al. (2012). Experimental and In Silico Modelling Analyses of the Gene Expression Pathway for Recombinant Antibody and By-Product Production in NS0 Cell Lines. PLoS ONE [Online] 7:e47422. Available at: http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0047422.
    Monoclonal antibodies are commercially important, high value biotherapeutic drugs used in the treatment of a variety of diseases. These complex molecules consist of two heavy chain and two light chain polypeptides covalently linked by disulphide bonds. They are usually expressed as recombinant proteins from cultured mammalian cells, which are capable of correctly modifying, folding and assembling the polypeptide chains into the native quaternary structure. Such recombinant cell lines often vary in the amounts of product produced and in the heterogeneity of the secreted products. The biological mechanisms of this variation are not fully defined. Here we have utilised experimental and modelling strategies to characterise and define the biology underpinning product heterogeneity in cell lines exhibiting varying antibody expression levels, and then experimentally validated these models. In undertaking these studies we applied and validated biochemical (rate-constant based) and engineering (nonlinear) models of antibody expression to experimental data from four NS0 cell lines with different IgG4 secretion rates. The models predict that export of the full antibody and its fragments are intrinsically linked, and cannot therefore be manipulated individually at the level of the secretory machinery. Instead, the models highlight strategies for the manipulation at the precursor species level to increase recombinant protein yields in both high and low producing cell lines. The models also highlight cell line specific limitations in the antibody expression pathway.
  • Chu, D., Barnes, D. and von der Haar, T. (2011). The role of tRNA and ribosome competition in coupling the expression of different mRNAs in Saccharomyces cerevisiae. Nucleic Acids Research [Online] 39:6705-6714. Available at: http://dx.doi.org/10.1093/nar/gkr300.
    Protein synthesis translates information from messenger RNAs into functional proteomes. Because of the finite nature of the resources required by the translational machinery, both the overall protein synthesis activity of a cell and activity on individual mRNAs are controlled by the allocation of limiting resources. Upon introduction of heterologous sequences into an organism—for example for the purposes of bioprocessing or synthetic biology—limiting resources may also become overstretched, thus negatively affecting both endogenous and heterologous gene expression. In this study, we present a mean-field model of translation in Saccharomyces cerevisiae for the investigation of two particular translational resources, namely ribosomes and aminoacylated tRNAs. We firstly use comparisons of experiments with heterologous sequences and simulations of the same conditions to calibrate our model, and then analyse the behaviour of the translational system in yeast upon introduction of different types of heterologous sequences. Our main findings are that: competition for ribosomes, rather than tRNAs, limits global translation in this organism; that tRNA aminoacylation levels exert, at most, weak control over translational activity; and that decoding speeds and codon adaptation exert strong control over local (mRNA specific) translation rates.
  • Merritt, G. et al. (2010). Decoding accuracy in eRF1 mutants and its correlation with pleiotropic quantitative traits in yeast. Nucleic acids research [Online] 38:5479-5492. Available at: http://dx.doi.org/10.1093/nar/gkq338.
    Translation termination in eukaryotes typically requires the decoding of one of three stop codons UAA, UAG or UGA by the eukaryotic release factor eRF1. The molecular mechanisms that allow eRF1 to decode either A or G in the second nucleotide, but to exclude UGG as a stop codon, are currently not well understood. Several models of stop codon recognition have been developed on the basis of evidence from mutagenesis studies, as well as studies on the evolutionary sequence conservation of eRF1. We show here that point mutants of Saccharomyces cerevisiae eRF1 display significant variability in their stop codon read-through phenotypes depending on the background genotype of the strain used, and that evolutionary conservation of amino acids in eRF1 is only a poor indicator of the functional importance of individual residues in translation termination. We further show that many phenotypes associated with eRF1 mutants are quantitatively unlinked with translation termination defects, suggesting that the evolutionary history of eRF1 was shaped by a complex set of molecular functions in addition to translation termination. We reassess current models of stop-codon recognition by eRF1 in the light of these new data.
  • Mead, E. et al. (2009). Identification of the limitations on recombinant gene expression in CHO cell lines with varying luciferase production rates. Biotechnology and bioengineering 102:1593-602.
    Mammalian cell lines are currently employed as one of the main cellular factories for the expression of recombinant protein-based drugs. The establishment of high-producing cell lines typically begins with a heterogeneous starter population of cells, from which the highest producing cells are selected via empirical approaches. This approach is time consuming, and is likely to encounter natural upper limits imposed by the inherent biology of the cell lines in question. In an attempt to understand both the nature of the variability in populations of cells transfected with recombinant protein encoding DNA and the natural mechanisms of productivity limitation, we developed protocols for the detailed investigation of gene expression pathways in such cell lines. This novel approach was then applied to a set of clonal CHOK1 cell lines producing recombinant luciferase with varying productivities. Our results show that the initial limitation in these cell lines is at the transcriptional level, however in the highest producing cell line post-translational mechanisms affecting both protein turnover and protein folding become severely limiting. The implications for the development of strategies to engineer cells for enhanced recombinant protein production levels are discussed.
  • Mead, E. et al. (2009). Identification of the limitations on recombinant gene expression in CHO cell lines with varying luciferase production rates. Biotechnology and Bioengineering [Online] 102:1593-1602. Available at: http://dx.doi.org/10.1002/bit.22201.
    Mammalian cell lines are currently employed as one of the main cellular factories for the expression of recombinant protein-based drugs. The establishment of high-producing cell lines typically begins with a heterogeneous starter population of cells, from which the highest producing cells are selected via empirical approaches. This approach is time consuming, and is likely to encounter natural upper limits imposed by the inherent biology of the cell lines in question. In an attempt to understand both the nature of the variability in populations of cells transfected with recombinant protein encoding DNA and the natural mechanisms of productivity limitation, we developed protocols for the detailed investigation of gene expression pathways in such cell lines. This novel approach was then applied to a set of clonal CHOK1 cell lines producing recombinant luciferase with varying productivities. Our results show that the initial limitation in these cell lines is at the transcriptional level, however in the highest producing cell line post-translational mechanisms affecting both protein turnover and protein folding become severely limiting. The implications for the development of strategies to engineer cells for enhanced recombinant protein production levels are discussed.
  • von der Haar, T. (2009). One for all? A viral protein supplants the mRNA cap-binding complex. Embo Journal [Online] 28:6-7. Available at: http://dx.doi.org/10.1038/emboj.2008.251.
    Modulation of the host cell’s translational machinery is a crucial part of viral infection strategies.Well-characterised mechanisms that aid viruses in manipulating translational activity include, for example, internal ribosomal entry sites, which allow viral RNA translation in the absence of some or many of the canonical host translation factors. New research shows that the nucleocapsid protein from a species of Hantavirus can replace several host cell translation factors in in vitro translation reactions, suggesting that hantaviruses may have evolved a novel strategy for modulating host cell translation in the form of a multifunctional translation factor.
  • Studte, P. et al. (2008). tRNA and protein methylase complexes mediate zymocin toxicity in yeast. Molecular Microbiology [Online] 69:1266-1277. Available at: http://dx.doi.org/10.1111/j.1365-2958.2008.06358.x.
    Modification of Saccharomyces cerevisiae tRNA anticodons at the wobble uridine (U34) position is required for tRNA cleavage by the zymocin tRNase killer toxin from Kluyveromyces lactis. Hence, U34 modification defects including lack of the U34 tRNA methyltransferase Trm9 protect against tRNA cleavage and zymocin. Using zymocin as a tool, we have identified toxin-resistant mutations in TRM9 that are likely to affect the U34 methylation reaction. Most strikingly, C-terminal truncations in Trm9 abolish interaction with Trm112, a protein shown to individually purify with Lys9 and two more methylases, Trm11 and Mtq2. Downregulation of a GAL1-TRM112 allele protects against zymocin whereas LYS9, TRM11 and MTQ2 are dosage suppressors of zymocin. Based on immune precipitation studies, the latter scenario correlates with competition for Trm112 and in excess, some of these Trm112 partners interfere with formation of the toxin-relevant Trm9.Trm112 complex. In contrast to trm11 Delta or lys9 Delta cells, trm112 Delta and mtq2 Delta null mutants are zymocin resistant. In line with the identified role that methylation of Sup45 by Mtq2 has for translation termination by the release factor dimer Sup45.Sup35, we observe that SUP45 overexpression and sup45 mutants suppress zymocin. Intriguingly, this suppression correlates with upregulated levels of tRNA species targeted by zymocin's tRNase activity.
  • von der Haar, T. (2008). A quantitative estimation of the global translational activity in logarithmically growing yeast cells. BMC Systems Biology [Online] 2. Available at: http://dx.doi.org/10.1186/1752-0509-2-87.
    BACKGROUND: Translation of messenger mRNAs makes significant contributions to the control of gene expression in all eukaryotes. Because translational control often involves fractional changes in translational activity, good quantitative descriptions of translational activity will be required to achieve a comprehensive understanding of this aspect of biology. Data on translational activity are difficult to generate experimentally under physiological conditions, however, translational activity as a parameter is in principle accessible through published genome-wide datasets. RESULTS: An examination of the accuracy of genome-wide expression datasets generated for Saccharomyces cerevisiae shows that the available datasets suffer from large random errors within studies as well as systematic shifts in reported values between studies, which make predictions of translational activity at the level of individual genes relatively inaccurate. In contrast, predictions of cell-wide translational activity are possible from such datasets with higher accuracy, and current datasets predict a production rate of about 13,000 proteins per haploid cell per second under fast growth conditions. This prediction is shown to be consistent with independently derived kinetic information on nucleotide exchange reactions that occur during translation, and on the ribosomal content of yeast cells. CONCLUSIONS: This study highlights some of the limitations in published genome-wide expression datasets, but also demonstrates a novel use for such datasets in examining global properties of cells. The global translational activity of yeast cells predicted in this study is a useful benchmark against which biochemical data on individual translation factor activities can be interpreted.
  • von der Haar, T. and Tuite, M. (2007). Regulated translational bypass of stop codons in yeast. Trends in Microbiology [Online] 15:78-86. Available at: http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6TD0-4MMFVM7-1&_user=125871&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_acct=C000010239&_version=1&_urlVersion=0&_userid=125871&md5=8405943db881e3df6b0707ef075a9c1e.
    Stop codons are used to signal the ribosome to terminate the decoding of an mRNA template. Recent studies on translation termination in the yeast Saccharomyces cerevisiale have not only enabled the identification of the key components of the termination machinery, but have also revealed several regulatory mechanisms that might enable the controlled synthesis of C-terminally extended polypeptides via stop-codon readthrough. These include both genetic and epigenetic mechanisms. Rather than being a translation 'error', stop-codon readthrough can have important effects on other cellular processes such as mRNA degradation and, in some cases, can confer a beneficial phenotype to the cell
  • von der Haar, T. et al. (2007). Development of a novel yeast cell-based system for studying the aggregation of Alzheimer's disease-associated A beta peptides in vivo. Neurodegenerative Diseases [Online] 4:136-147. Available at: http://dx.doi.org/10.1159/000101838.
    Alzheimer's disease is the most common neurodegenerative disease, affecting -50% of humans by age 85. The disease process is associated with aggregation of the AP peptides, short 39-43 residue peptides generated through endoproteolytic cleavage of the Alzheimer's precursor protein. While the process of aggregation of purified AP peptides in vitro is beginning to be well understood, little is known about this process in vivo. In the present study, we use the yeast Saccharomyces cerevisiae as a model system for studying A beta-mediated aggregation in an organism in vivo. One of this yeast's endogenous prions, Sup35/[PSI+] loses the ability to aggregate when the prion-forming domain of this protein is deleted. We show that insertion of AP pepticle sequences in place of the original prion domain of this protein restores its ability to aggregate. However, the aggregates are qualitatively different from [PSI+] prions in their sensitivity to detergents and in their requirements on transacting factors that are normally needed for [PSI+] propagation. We conclude that we have established a useful new tool for studying the aggregation of AP peptides in an organism in vivo.
  • Gilbert, R. et al. (2007). Reconfiguration of yeast 40S ribosomal subunit domains by the translation initiation multifactor complex. Proceedings of the National Academy of Sciences of the United States of America [Online] 104:5788-5793. Available at: http://dx.doi.org/10.1073/pnas.0606880104.
    In the process of protein synthesis, the small (40S) subunit of the eukaryotic ribosome is recruited to the capped 5' end of the mRNA, from which point it scans along the 5' untranslated region in search of a start codon. However, the 40S subunit alone is not capable of functional association with cellular mRNA species; it has to be prepared for the recruitment and scanning steps by interactions with a group of eukaryotic initiation factors (eIFs). In budding yeast, an important subset of these factors (1, 2, 3, and 5) can form a multifactor complex (MFC). Here, we describe cryo-EM reconstructions of the 40S subunit, of the MFC, and of 40S complexes with MFC factors plus eIF1A. These studies reveal the positioning of the core MFC on the 40S subunit, and show how eIF-binding induces mobility in the head and platform and reconfigures the head–platform–body relationship. This is expected to increase the accessibility of the mRNA channel, thus enabling the 40S subunit to convert to a recruitment-competent state.
  • von der Haar, T. (2007). Optimized protein extraction for quantitative proteomics of yeasts. PLoS ONE [Online] 2:e1078. Available at: http://dx.doi.org/10.1371/journal.pone.0001078.
    BACKGROUND: The absolute quantification of intracellular protein levels is technically demanding, but has recently become more prominent because novel approaches like systems biology and metabolic control analysis require knowledge of these parameters. Current protocols for the extraction of proteins from yeast cells are likely to introduce artifacts into quantification procedures because of incomplete or selective extraction. PRINCIPAL FINDINGS: We have developed a novel procedure for protein extraction from S. cerevisiae based on chemical lysis and simultaneous solubilization in SDS and urea, which can extract the great majority of proteins to apparent completeness. The procedure can be used for different Saccharomyces yeast species and varying growth conditions, is suitable for high-throughput extraction in a 96-well format, and the resulting extracts can easily be post-processed for use in non-SDS compatible procedures like 2D gel electrophoresis. CONCLUSIONS: An improved method for quantitative protein extraction has been developed that removes some of the sources of artefacts in quantitative proteomics experiments, while at the same time allowing novel types of applications.
  • von der Haar, T. et al. (2006). Folding transitions during assembly of the eukaryotic mRNA cap-binding complex. Journal of Molecular Biology [Online] 356:982-992. Available at: http://dx.doi.org/10.1016/j.jmb.2005.12.034.
    The cap-binding protein eIF4E is the first in a chain of translation initiation factors that recruit 40S ribosomal subunits to the 5' end of eukaryotic mRNA. During cap-dependent translation, this protein binds to the 5'-terminal m(7)Gppp cap of the mRNA, as well as to the adaptor protein eIF4G. The latter then interacts with small ribosomal subunit-bound proteins, thereby promoting the mRNA recruitment process. Here, we show apo-eIF4E to be a protein that contains extensive unstructured regions, which are induced to fold upon recognition of the cap structure. Binding of eIF4G to apo-eIF4E likewise induces folding of the protein into a state that is similar to, but not identical with, that of cap-bound eIF4E. At the same time, binding of each of the binding partners of eIF4E modulates the kinetics with which it interacts with the other partner. We present structural, kinetic and mutagenesis data that allow us to deduce some of the detailed folding transitions that take place during the eIF4E interactions.
  • von der Haar, T. et al. (2004). The mRNA cap-binding protein eIF4E in post-transcriptional gene expression. Nature Structural and Molecular Biology [Online] 11:503-511. Available at: http://dx.doi.org/10.1038/nsmb779.
    Eukaryotic initiation factor 4E (eIF4E) has central roles in the control of several aspects of post-transcriptional gene expression and thereby affects developmental processes. It is also implicated in human diseases. This review explores the relationship between structural, biochemical and biophysical aspects of eIF4E and its function in vivo, including both long-established roles in translation and newly emerging ones in nuclear export and mRNA decay pathways.
  • He, H. and von der Haar, T. (2003). The yeast eukaryotic initiation factor 4G (eIF4G) HEAT domain interacts with eIF1 and eIF5 and is involved in stringent AUG selection. Molecular and Cellular Biology [Online] 23:5431-5445. Available at: http://dx.doi.org/10.1128/MCB.23.15.5431-5445.2003.
    Eukaryotic initiation factor 4G (eIF4G) promotes mRNA recruitment to the ribosome by binding to the mRNA cap- and poly(A) tail-binding proteins eIF4E and Pap1p. eIF4G also binds eIF4A at a distinct HEAT domain composed of five stacks of antiparallel alpha-helices. The role of eIF4G in the later steps of initiation, such as scanning and AUG recognition, has not been defined. Here we show that the entire HEAT domain and flanking residues of Saccharomyces cerevisiae eIF4G2 are required for the optimal interaction with the AUG recognition factors eIF5 and eIF1. eIF1 binds simultaneously to eIF4G and eIF3c in vitro, as shown previously for the C-terminal domain of eIF5. In vivo, co-overexpression of eIF1 or eIF5 reverses the genetic suppression of an eIF4G HEAT domain Ts(-) mutation by eIF4A overexpression. In addition, excess eIF1 inhibits growth of a second eIF4G mutant defective in eIF4E binding, which was also reversed by co-overexpression of eIF4A. Interestingly, excess eIF1 carrying the sui1-1 mutation, known to relax the accuracy of start site selection, did not inhibit the growth of the eIF4G mutant, and sui1-1 reduced the interaction between eIF4G and eIF1 in vitro. Moreover, a HEAT domain mutation altering eIF4G moderately enhances translation from a non-AUG codon. These results strongly suggest that the binding of the eIF4G HEAT domain to eIF1 and eIF5 is important for maintaining the integrity of the scanning ribosomal preinitiation complex.
  • von der Haar, T. and McCarthy, J. (2003). Studying the assembly of multicomponent protein and ribonucleoprotein complexes using surface plasmon resonance. Methods 29:167-174.
    The assembly of large macromolecular complexes is an important aspect of cellular organization and metabolism. Interactions involving such complexes in principle follow the same rules as the interactions between single proteins or other macromolecules and can therefore be investigated using similar approaches. We have developed protocols employing standard surface plasmon resonance technology that allow the investigation of interactions involving complex macromolecular structures. The principal experimental challenges arise from the possibility of parallel reactions where partially assembled or dissociated subcomplexes form a significant proportion of the molecule population and from an increased likelihood of unspecific binding events owing to the larger surface and statistically higher number of charged areas on multisubunit assemblies. Ways to experimentally avoid or, where this is not possible, to control for these complications are discussed.
  • Gross, J. et al. (2003). Ribosome loading onto the mRNA cap is driven by conformational coupling between eIF4G and eIF4E. Cell [Online] 115:739-750. Available at: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=14675538.
    The eukaryotic initiation factor 4G (eIF4G) is the core of a multicomponent switch controlling gene expression at the level of translation initiation. It interacts with the small ribosomal subunit interacting protein, eIF3, and the eIF4E/cap-mRNA complex in order to load the ribosome onto mRNA during cap-dependent translation. We describe the solution structure of the complex between yeast eIF4E/cap and eIF4G (393-490). Binding triggers a coupled folding transition of eIF4G (393-490) and the eIF4E N terminus resulting in a molecular bracelet whereby eIF4G (393-490) forms a right-handed helical ring that wraps around the N terminus of eIF4E. Cofolding allosterically enhances association of eIF4E with the cap and is required for maintenance of optimal growth and polysome distributions in vivo. Our data explain how mRNA, eIF4E, and eIF4G exists as a stable mRNP that may facilitate multiple rounds of ribosomal loading during translation initiation, a key determinant in the overall rate of protein synthesis.
  • von der Haar, T. et al. (2002). Translation initiation and surface plasmon resonance: new technology applied to old questions. Biochemical Society Transactions 30:155-162.
    Translation initiation in eukaryotes is a complicated process involving some of the largest cellular structures, the ribosomes, together with approx. 11 initiation factors, and a poorly characterized set of other proteins. The concerted action of all these components ultimately results in the formation of an 80 S ribosomal complex on the AUG codon of an mRNA, which is competent to start polypeptide production. In this brief overview, we describe the strategies developed by our laboratory to apply surface plasmon resonance (SPR)-based technology to the problem of elucidating kinetic aspects of substeps within the translation-initiation reaction. We then review how other groups have used similar SPR-based techniques to study related interactions.
  • von der Haar, T. and McCarthy, J. (2002). Intracellular translation initiation factor levels in Saccharomyces cerevisiae and their role in cap-complex function. Molecular Microbiology [Online] 46:531-544. Available at: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=12406227.
    Knowledge of the balance of activities of eukaryotic initiation factors (eIFs) is critical to our understanding of the mechanisms underlying translational control. We have therefore estimated the intracellular levels of 11 eIFs in logarithmically growing cells of Saccharomyces cerevisiae using polyclonal antibodies raised in rabbits against recombinant proteins. Those factors involved in 43S complex formation occur at levels comparable (i.e. within a 0.5- to 2.0-fold range) to those published for ribosomes. In contrast, the subunits of the cap-binding complex eIF4F showed considerable variation in their abundance. The helicase eIF4A was the most abundant eIF of the yeast cell, followed by eIF4E at multiple copies per ribosome, and eIF4B at approximately one copy per ribosome. The adaptor protein eIF4G was the least abundant of the eIF4 factors, with a copy number per cell that is substoichiometric to the ribosome and similar to the abundance of mRNA. The observed excess of eIF4E over its functional partner eIF4G is not strictly required during exponential growth: at eIF4E levels artificially reduced to 30% of those in wild-type yeast, growth rates and the capacity for general protein synthesis are only minimally affected. This demonstrates that eIF4E does not exercise a higher level of rate control over translation than other eIFs. However, other features of the yeast life cycle, such as the control of cell size, are more sensitive to changes in eIF4E abundance. Overall, these data constitute an important basis for developing a quantitative model of the workings of the eukaryotic translation apparatus.

Book section

  • Tuite, M. and von der Haar, T. (2016). Transfer RNA in Decoding and the Wobble Hypothesis. in: eLS. Wiley, pp. 1-7. Available at: http://doi.org/10.1002/9780470015902.a0001497.pub2.
    Translation of the genetic code stored in messenger ribonucleic acid (RNA) requires significantly fewer transfer RNAs (35–45) than there are codons (61, amino acid specifying). This is achieved through an increased flexibility in the allowable base-pair interactions between the messenger RNA and the transfer RNA involving the third position of the codon and the first position of the corresponding anticodon. The rules governing this RNA:RNA interaction were originally summarised in Crick's ‘wobble hypothesis’. Covalent modification of the first base of an anticodon of a transfer RNA can profoundly affect the degree of flexibility in its base-pairing potential by either extending or restricting such interactions. Recent studies suggest that the rate at which a codon is processed by the ribosome is influenced by whether or not decoding of that codon is via wobble base interactions. Yet, in spite of this flexibility and different rates of processing, decoding by transfer RNAs is achieved with considerable accuracy.
  • von der Haar, T. and Valasek, L. (2014). mRNA Translation: Fungal Variations on a Eukaryotic Theme. in: Sesma, A. and von der Haar, T. eds. Fungal RNA Biology. Springer International Publishing, pp. 113-134. Available at: http://dx.doi.org/10.1007/978-3-319-05687-6_5.
    The accurate transfer of information from a nucleotide-based code to a protein-based one is at the heart of all life processes. The actual information transfer occurs during protein synthesis or translation, and is catalysed by ribosomes, supported by a large host of additional protein activities—the translation factors. This chapter reviews how the different eukaryotic initiation, elongation and termination factors assist the ribosome in establishing appropriate contacts with mRNAs during translation initiation, decode the genetic code during translation elongation, and finally release the newly made polypeptide and reuse the ribosomes during the termination and recycling phases.
  • von der Haar, T., Jossé, L. and Byrne, L. (2007). Reporter genes and their uses in studying yeast gene expression. in: Yeast gene analysis, second edition. San Diego, US: Elsevier Academic Press, pp. 165-188.
  • McCarthy, J., Marsden, S. and von der Haar, T. (2007). Biophysical studies of the translation initiation pathway using immobilised mRNA analogues. in: Lorsch, J. ed. Translation Initiation: Reconstituted Systems and Biophysical Methods. Elsevier, pp. 247-264. Available at: http://dx.doi.org/10.1016/S0076-6879(07)30010-4.
    A growing number of biophysical techniques use immobilized reactants for the quantitative study of macromolecular reactions. Examples of such approaches include surface plasmon resonance, atomic force microscopy, total reflection fluorescence microscopy, and others. Some of these methods have already been adapted for work with immobilized RNAs, thus making them available for the study of many reactions relevant to translation. Published examples include the study of kinetic parameters of protein/RNA interactions and the effect of helicases on RNA secondary structure. The common denominator of all of these techniques is the necessity to immobilize RNA molecules in a functional state on solid supports. In this chapter, we describe a number of approaches by which such immobilization can be achieved, followed by two specific examples for applications that use immobilized RNAs.

Edited book

  • Sesma, A. and von der Haar, T. eds. (2014). Fungal RNA Biology. [Online]. Springer International Publishing. Available at: http://www.springer.com/gb/book/9783319056869.
    This book presents an overview of the RNA networks controlling gene expression in fungi highlighting the remaining questions and future challenges in this area.

    It covers several aspects of the RNA-mediated mechanisms that regulate gene expression in model yeasts and filamentous fungi, organisms of great importance for industry, medicine and agriculture. It is estimated that there are more than one million fungal species on the Earth. Despite their diversity (saprophytic, parasitic and mutualistic), fungi share common features distinctive from plants and animals and have been grouped taxonomically as an independent eukaryotic kingdom. In this book, 15 chapters written by experts in their fields cover the RNA-dependent processes that take place in a fungal cell ranging from formation of coding and non-coding RNAs to mRNA translation, ribosomal RNA biogenesis, gene silencing, RNA editing and epigenetic regulation.

Thesis

  • Ji, H. (2015). Fibrinolytic Regulation of Pulmonary Epithelial Sodium Channels: a Critical Review.
    Luminal fluid homeostasis in the respiratory system is crucial to maintain the gas-
    blood exchange in normal lungs and mucociliary clearance in the airways. Epithelial
    sodium channels (ENaC) govern ~70% of alveolar fluid clearance. Four ENaC subunits
    have been cloned, namely, ?, ?, ?, and ? ENaC subunits in mammalian cells. This
    critical review focuses on the expression and function of ENaC in human and murine
    lungs, and the post-translational regulation by fibrinolysins. Nebulized urokinase was
    intratracheally delivered for clinical models of lung injury with unknown mechanisms.
    The central hypothesis is that proteolytically cleaved ENaC channels composed of four
    subunits are essential pathways to maintain fluid homeostasis in the airspaces, and that
    fibrinolysins are potential pharmaceutical ENaC activators to resolve edema fluid. This
    hypothesis is strongly supported by our following observations: 1) ? ENaC is expressed
    in the apical membrane of human lung epithelial cells; 2) ? ENaC physically interacts
    with the other three ENaC counterparts; 3) the features of ??? ENaC channels are
    conferred by ? ENaC; 4) urokinase activates ENaC activity; 5) urokinase deficiency is
    associated with a markedly distressed pulmonary ENaC function in vivo; 6) ? ENaC is
    proteolytically cleaved by urokinase; 7) urokinase augments the density of opening
    channels at the cell surface; and 8) urokinase extends opening time of ENaC channels
    to the most extent. Our integrated publications laid the groundwork for an innovative
    concept of pulmonary transepithelial fluid clearance in both normal and diseased lungs.

Forthcoming

  • Deng, Y. et al. (2019). Hidden patterns of codon usage bias across kingdoms. BioRxiv [Online]. Available at: https://dx.doi.org/10.1101/478016.
    The genetic code encodes 20 amino acids using 64 nucleotide triplets or codons. 18 of the 20 amino acids are encoded by multiple synonymous codons which are used in organismal genomes in a biased fashion. Codon bias arises because evolutionary selection favours particular nucleotide sequences over others encoding the same amino acid sequence. Despite many existing hypotheses, there is no current consensus on what the evolutionary drivers are. Using ideas from stochastic thermodynamics we derive from first principles a mathematical model describing the statistics of codon usage bias and apply it to extensive genomic data. Our main conclusions include the following findings: (1) Codon usage cannot be explained solely by selection pressures that act on the genome-wide frequency of codons, but also includes pressures that act at the level of individual genes. (2) Codon usage is not only biased in the usage frequency of nucleotide triplets but also in how they are distributed across mRNAs. (3) A new model-based measure of codon usage bias that extends existing measures by taking into account both codon frequency and codon distribution reveals distinct, amino acid specific patterns of selection in distinct branches of the tree of life.
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