TY - JOUR A1 - Adamla, Frauke A1 - Ignatova, Zoya T1 - Somatic expression of unc-54 and vha-6 mRNAs declines but not pan-neuronal rgef-1 and unc-119 expression in aging Caenorhabditis elegans JF - Scientific reports N2 - Aging is a highly controlled biological process characterized by a progressive deterioration of various cellular activities. One of several hallmarks of aging describes a link to transcriptional alteration, suggesting that it may impact the steady-state mRNA levels. We analyzed the mRNA steady-state levels of polyCAG-encoding transgenes and endogenous genes under the control of well-characterized promoters for intestinal (vha-6), muscular (unc-54, unc-15) and pan-neuronal (rgef-1, unc-119) expression in the nematode Caenorhabditis elegans. We find that there is not a uniform change in transcriptional profile in aging, but rather a tissue-specific difference in the mRNA levels of these genes. While levels of mRNA in the intestine (vha-6) and muscular (unc-54, unc-15) cells decline with age, pan-neuronal tissue shows more stable mRNA expression (rgef-1, unc-119) which even slightly increases with the age of the animals. Our data on the variations in the mRNA abundance from exemplary cases of endogenous and transgenic gene expression contribute to the emerging evidence for tissue-specific variations in the aging process. Y1 - 2015 U6 - https://doi.org/10.1038/srep10692 SN - 2045-2322 VL - 5 PB - Nature Publ. Group CY - London ER - TY - JOUR A1 - Avcilar-Kucukgoze, Irem A1 - Bartholomäus, Alexander A1 - Varela, Juan A. Cordero A1 - Kaml, Robert Franz-Xaver A1 - Neubauer, Peter A1 - Budisa, Nediljko A1 - Ignatova, Zoya T1 - Discharging tRNAs: a tug of war between translation and detoxification in Escherichia coli JF - Nucleic acids research N2 - Translation is a central cellular process and is optimized for speed and fidelity. The speed of translation of a single codon depends on the concentration of aminoacyl-tRNAs. Here, we used microarray-based approaches to analyze the charging levels of tRNAs in Escherichia coli growing at different growth rates. Strikingly, we observed a non-uniform aminoacylation of tRNAs in complex media. In contrast, in minimal medium, the level of aminoacyl-tRNAs is more uniform and rises to approximately 60%. Particularly, the charging level of tRNA(Ser), tRNA(Cys), tRNA(Thr) and tRNA(His) is below 50% in complex medium and their aminoacylation levels mirror the degree that amino acids inhibit growth when individually added to minimal medium. Serine is among the most toxic amino acids for bacteria and tRNAs(Ser) exhibit the lowest charging levels, below 10%, at high growth rate although intracellular serine concentration is plentiful. As a result some serine codons are among the most slowly translated codons. A large fraction of the serine is most likely degraded by L-serine-deaminase, which competes with the seryl-tRNA-synthetase that charges the tRNAs(Ser). These results indicate that the level of aminoacylation in complex media might be a competition between charging for translation and degradation of amino acids that inhibit growth. Y1 - 2016 U6 - https://doi.org/10.1093/nar/gkw697 SN - 0305-1048 SN - 1362-4962 VL - 44 SP - 8324 EP - 8334 PB - Oxford Univ. Press CY - Oxford ER - TY - JOUR A1 - Bartholomäus, Alexander A1 - Fedyunin, Ivan A1 - Feist, Peter A1 - Sin, Celine A1 - Zhang, Gong A1 - Valleriani, Angelo A1 - Ignatova, Zoya T1 - Bacteria differently regulate mRNA abundance to specifically respond to various stresses JF - Geology N2 - Environmental stress is detrimental to cell viability and requires an adequate reprogramming of cellular activities to maximize cell survival. We present a global analysis of the response of Escherichia coli to acute heat and osmotic stress. We combine deep sequencing of total mRNA and ribosome-protected fragments to provide a genome-wide map of the stress response at transcriptional and translational levels. For each type of stress, we observe a unique subset of genes that shape the stress-specific response. Upon temperature upshift, mRNAs with reduced folding stability up-and downstream of the start codon, and thus with more accessible initiation regions, are translationally favoured. Conversely, osmotic upshift causes a global reduction of highly translated transcripts with high copy numbers, allowing reallocation of translation resources to not degraded and newly synthesized mRNAs. KW - transcription KW - translation KW - deep sequencing KW - Escherichia coli KW - copy numbers Y1 - 2016 U6 - https://doi.org/10.1098/rsta.2015.0069 SN - 1364-503X SN - 1471-2962 VL - 374 PB - Royal Society CY - London ER - TY - GEN A1 - Bentele, Kajetan A1 - Saffert, Paul A1 - Rauscher, Robert A1 - Ignatova, Zoya A1 - Bluethgen, Nils T1 - Efficient translation initiation dictates codon usage at gene start T2 - Postprints der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe N2 - The genetic code is degenerate; thus, protein evolution does not uniquely determine the coding sequence. One of the puzzles in evolutionary genetics is therefore to uncover evolutionary driving forces that result in specific codon choice. In many bacteria, the first 5-10 codons of protein-coding genes are often codons that are less frequently used in the rest of the genome, an effect that has been argued to arise from selection for slowed early elongation to reduce ribosome traffic jams. However, genome analysis across many species has demonstrated that the region shows reduced mRNA folding consistent with pressure for efficient translation initiation. This raises the possibility that unusual codon usage is a side effect of selection for reduced mRNA structure. Here we discriminate between these two competing hypotheses, and show that in bacteria selection favours codons that reduce mRNA folding around the translation start, regardless of whether these codons are frequent or rare. Experiments confirm that primarily mRNA structure, and not codon usage, at the beginning of genes determines the translation rate. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 912 KW - codon usage KW - mRNA structure KW - translation Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-441337 SN - 1866-8372 IS - 912 ER - TY - JOUR A1 - Bentele, Kajetan A1 - Saffert, Paul A1 - Rauscher, Robert A1 - Ignatova, Zoya A1 - Bluethgen, Nils T1 - Efficient translation initiation dictates codon usage at gene start JF - Molecular systems biology N2 - The genetic code is degenerate; thus, protein evolution does not uniquely determine the coding sequence. One of the puzzles in evolutionary genetics is therefore to uncover evolutionary driving forces that result in specific codon choice. In many bacteria, the first 5-10 codons of protein-coding genes are often codons that are less frequently used in the rest of the genome, an effect that has been argued to arise from selection for slowed early elongation to reduce ribosome traffic jams. However, genome analysis across many species has demonstrated that the region shows reduced mRNA folding consistent with pressure for efficient translation initiation. This raises the possibility that unusual codon usage is a side effect of selection for reduced mRNA structure. Here we discriminate between these two competing hypotheses, and show that in bacteria selection favours codons that reduce mRNA folding around the translation start, regardless of whether these codons are frequent or rare. Experiments confirm that primarily mRNA structure, and not codon usage, at the beginning of genes determines the translation rate. KW - codon usage KW - mRNA structure KW - translation Y1 - 2013 U6 - https://doi.org/10.1038/msb.2013.32 SN - 1744-4292 VL - 9 IS - 6 PB - Nature Publ. Group CY - New York ER - TY - JOUR A1 - Borwankar, Tejas A1 - Roethlein, Christoph A1 - Zhang, Gong A1 - Techen, Anne A1 - Dosche, Carsten A1 - Ignatova, Zoya T1 - Natural osmolytes remodel the aggregation pathway of mutant huntingtin exon 1 JF - Biochemistry N2 - In response to stress small organic compounds termed osmolytes are ubiquitously accumulated in all cell types to regulate the intracellular solvent quality and to counteract the deleterious effect on the stability and function of cellular proteins. Given the evidence that destabilization of the native state of a protein either by mutation or by environmental changes triggers the aggregation in the neurodegenerative pathologies, the modulation of the intracellular solute composition with osmolytes is an attractive strategy to stabilize an aggregating protein. Here we report the effect of three natural osmolytes on the in vivo and in vitro aggregation landscape of huntingtin exon 1 implicated in the Huntington's disease. Trimethylamine N-oxide (TMAO) and proline redirect amyloid fibrillogenesis of the pathological huntingtin exon 1 to nonamyloidogenic amorphous assemblies via two dissimilar molecular mechanisms. TMAO causes a rapid formation of bulky amorphous aggregates with minimally exposed surface area, whereas proline solubilizes the monomer and suppresses the accumulation of early transient aggregates. Conversely, glycine betaine enhances fibrillization in a fashion reminiscent of the genesis of functional amyloids. Strikingly, none of the natural osmolytes can completely abrogate the aggregate formation; however, they redirect the amyloidogenesis into alternative, nontoxic aggregate species. Our study reveals new insights into the complex interactions of osmoprotectants with polyQaggregates. Y1 - 2011 U6 - https://doi.org/10.1021/bi1018368 SN - 0006-2960 VL - 50 IS - 12 SP - 2048 EP - 2060 PB - American Chemical Society CY - Washington ER - TY - JOUR A1 - Czech, Andreas A1 - Fedyunin, Ivan A1 - Zhang, Gong A1 - Ignatova, Zoya T1 - Silent mutations in sight : co-variations in tRNA abundance as a key to unravel consequences of silent mutations N2 - Mutations that alter the amino acid sequence are known to potentially exert deleterious effects on protein function, whereas substitutions of nucleotides without amino acid change are assumed to be neutral for the protein's functionality. However, cumulative evidence suggests that synonymous substitutions might also induce phenotypic variability by affecting splicing accuracy, translation fidelity, and conformation and function of proteins. tRNA isoacceptors mediate the translation of codons to amino acids, and asymmetric tRNA abundance causes variations in the rate of translation of each single triplet. Consequently, the effect of a silent point mutation in the coding region could be significant due to differential abundances of the cognate tRNA(s), emphasizing the importance of precise assessment of tRNA composition. Here, we provide an overview of the methods used to quantitatively determine the concentrations of tRNA species and discuss synonymous mutations in the context of tRNA composition of the cell, thus providing a new twist on the detrimental impact of the silent mutations. Y1 - 2010 UR - http://www.rsc.org/Publishing/Journals/mb/index.asp U6 - https://doi.org/10.1039/C004796c SN - 1742-206X ER - TY - JOUR A1 - Czech, Andreas A1 - Wende, Sandra A1 - Moerl, Mario A1 - Pan, Tao A1 - Ignatova, Zoya T1 - Reversible and rapid transfer-RNA deactivation as a mechanism of translational repression in stress JF - PLoS Genetics : a peer-reviewed, open-access journal N2 - Stress-induced changes of gene expression are crucial for survival of eukaryotic cells. Regulation at the level of translation provides the necessary plasticity for immediate changes of cellular activities and protein levels. In this study, we demonstrate that exposure to oxidative stress results in a quick repression of translation by deactivation of the aminoacylends of all transfer-RNA (tRNA). An oxidative-stress activated nuclease, angiogenin, cleaves first within the conserved single-stranded 3'-CCA termini of all tRNAs, thereby blocking their use in translation. This CCA deactivation is reversible and quickly repairable by the CCA-adding enzyme [ATP(CTP): tRNA nucleotidyltransferase]. Through this mechanism the eukaryotic cell dynamically represses and reactivates translation at low metabolic costs. Y1 - 2013 U6 - https://doi.org/10.1371/journal.pgen.1003767 SN - 1553-7404 VL - 9 IS - 8 PB - PLoS CY - San Fransisco ER - TY - GEN A1 - Del Campo, Cristian A1 - Bartholomäus, Alexander A1 - Fedyunin, Ivan A1 - Ignatova, Zoya T1 - Secondary Structure across the Bacterial Transcriptome Reveals Versatile Roles in mRNA Regulation and Function T2 - Postprints der Universität Potsdam : Mathematisch Naturwissenschaftliche Reihe N2 - Messenger RNA acts as an informational molecule between DNA and translating ribosomes. Emerging evidence places mRNA in central cellular processes beyond its major function as informational entity. Although individual examples show that specific structural features of mRNA regulate translation and transcript stability, their role and function throughout the bacterial transcriptome remains unknown. Combining three sequencing approaches to provide a high resolution view of global mRNA secondary structure, translation efficiency and mRNA abundance, we unraveled structural features in E. coli mRNA with implications in translation and mRNA degradation. A poorly structured site upstream of the coding sequence serves as an additional unspecific binding site of the ribosomes and the degree of its secondary structure propensity negatively correlates with gene expression. Secondary structures within coding sequences are highly dynamic and influence translation only within a very small subset of positions. A secondary structure upstream of the stop codon is enriched in genes terminated by UAA codon with likely implications in translation termination. The global analysis further substantiates a common recognition signature of RNase E to initiate endonucleolytic cleavage. This work determines for the first time the E. coli RNA structurome, highlighting the contribution of mRNA secondary structure as a direct effector of a variety of processes, including translation and mRNA degradation. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 520 KW - Escherichia coli KW - in vivo KW - translation initiation KW - crystal-structure KW - single ribosomes KW - gene-expression KW - global analysis KW - codon usage KW - E-cleavage KW - genome Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-409662 SN - 1866-8372 IS - 520 ER - TY - JOUR A1 - Del Campo, Cristian A1 - Bartholomäus, Alexander A1 - Fedyunin, Ivan A1 - Ignatova, Zoya T1 - Secondary Structure across the Bacterial Transcriptome Reveals Versatile Roles in mRNA Regulation and Function JF - PLoS Genetics : a peer-reviewed, open-access journal N2 - Messenger RNA acts as an informational molecule between DNA and translating ribosomes. Emerging evidence places mRNA in central cellular processes beyond its major function as informational entity. Although individual examples show that specific structural features of mRNA regulate translation and transcript stability, their role and function throughout the bacterial transcriptome remains unknown. Combining three sequencing approaches to provide a high resolution view of global mRNA secondary structure, translation efficiency and mRNA abundance, we unraveled structural features in E. coli mRNA with implications in translation and mRNA degradation. A poorly structured site upstream of the coding sequence serves as an additional unspecific binding site of the ribosomes and the degree of its secondary structure propensity negatively correlates with gene expression. Secondary structures within coding sequences are highly dynamic and influence translation only within a very small subset of positions. A secondary structure upstream of the stop codon is enriched in genes terminated by UAA codon with likely implications in translation termination. The global analysis further substantiates a common recognition signature of RNase E to initiate endonucleolytic cleavage. This work determines for the first time the E. coli RNA structurome, highlighting the contribution of mRNA secondary structure as a direct effector of a variety of processes, including translation and mRNA degradation. Y1 - 2015 U6 - https://doi.org/10.1371/journal.pgen.1005613 SN - 1553-7404 VL - 11 IS - 10 PB - PLoS CY - San Fransisco ER - TY - JOUR A1 - Fedyunin, Ivan A1 - Lehnhardt, Lothar A1 - Böhmer, Nadine A1 - Kaufmann, Paul A1 - Zhang, Gong A1 - Ignatova, Zoya T1 - tRNA concentration fine tunes protein solubility JF - FEBS letters : the journal for rapid publication of short reports in molecular biosciences N2 - Clusters of codons pairing to low-abundance tRNAs synchronize the translation with co-translational folding of single domains in multidomain proteins. Although proven with some examples, the impact of the ribosomal speed on the folding and solubility on a global, cell-wide level remains elusive. Here we show that upregulation of three low-abundance tRNAs in Escherichia coil increased the aggregation propensity of several cellular proteins as a result of an accelerated elongation rate. Intriguingly, alterations in the concentration of the natural tRNA pool compromised the solubility of various chaperones consequently rendering the solubility of some chaperone-dependent proteins. KW - Protein translation KW - Protein misfolding KW - tRNA KW - E. coli Y1 - 2012 U6 - https://doi.org/10.1016/j.febslet.2012.07.012 SN - 0014-5793 VL - 586 IS - 19 SP - 3336 EP - 3340 PB - Elsevier CY - Amsterdam ER - TY - CHAP A1 - Ferrolino, Mylene A1 - Zhuravleva, Anastasia A1 - Ignatova, Zoya A1 - Gierasch, Lila T1 - Exploring In vitro and in vivo aggregation of a beta-Clam protein T2 - Protein science : a publication of the Protein Society Y1 - 2012 SN - 0961-8368 VL - 21 IS - 2 SP - 89 EP - 89 PB - Wiley-Blackwell CY - Hoboken ER - TY - JOUR A1 - Girstmair, Hannah A1 - Saffert, Paul A1 - Rode, Sascha A1 - Czech, Andreas A1 - Holland, Gudrun A1 - Bannert, Norbert A1 - Ignatova, Zoya T1 - Depletion of Cognate Charged Transfer RNA Causes Translational Frameshifting within the Expanded CAG Stretch in Huntingtin JF - Cell reports N2 - Huntington disease (HD), a dominantly inherited neurodegenerative disorder caused by the expansion of a CAG-encoded polyglutamine (polyQ) repeat in huntingtin (Htt), displays a highly heterogeneous etiopathology and disease onset. Here, we show that the translation of expanded CAG repeats in mutant Htt exon 1 leads to a depletion of charged glutaminyl-transfer RNA (tRNA) Gln-CUG that pairs exclusively to the CAG codon. This results in translational frameshifting and the generation of various transframe-encoded species that differently modulate the conformational switch to nucleate fibrillization of the parental polyQ protein. Intriguingly, the frameshifting frequency varies strongly among different cell lines and is higher in cells with intrinsically lower concentrations of tRNA Gln-CUG. The concentration of tRNA Gln-CUG also differs among different brain areas in the mouse. We propose that translational frameshifting may act as a significant disease modifier that contributes to the cell-selective neurotoxicity and disease course heterogeneity of HD on both cellular and individual levels. Y1 - 2013 U6 - https://doi.org/10.1016/j.celrep.2012.12.019 SN - 2211-1247 VL - 3 IS - 1 SP - 148 EP - 159 PB - Cell Press CY - Cambridge ER - TY - JOUR A1 - Gorochowski, Thomas E. A1 - Aycilar-Kucukgoze, Irem A1 - Bovenberg, Roel A. L. A1 - Roubos, Johannes A. A1 - Ignatova, Zoya T1 - A Minimal Model of Ribosome Allocation Dynamics Captures Trade-offs in Expression between Endogenous and Synthetic Genes JF - ACS synthetic biology N2 - Cells contain a finite set of resources that must be distributed across many processes to ensure survival. Among them, the largest proportion of cellular resources is dedicated to protein translation. Synthetic biology often exploits these resources in executing orthogonal genetic circuits, yet the burden this places on the cell is rarely considered. Here, we develop a minimal model of ribosome allocation dynamics capturing the demands on translation when expressing a synthetic construct together with endogenous genes required for the maintenance of cell physiology. Critically, it contains three key variables related to design parameters of the synthetic construct covering transcript abundance, translation initiation rate, and elongation time. We show that model-predicted changes in ribosome allocation closely match experimental shifts in synthetic protein expression rate and cellular growth. Intriguingly, the model is also able to accurately infer transcript levels and translation times after further exposure to additional ambient stress. Our results demonstrate that a simple model of resource allocation faithfully captures the redistribution of protein synthesis resources when faced with the burden of synthetic gene expression and environmental stress. The tractable nature of the model makes it a versatile tool for exploring the guiding principles of efficient heterologous expression and the indirect interactions that can arise between synthetic circuits and their host chassis because of competition for shared translational resources. KW - protein biosynthesis KW - translation KW - synthetic biology KW - systems biology Y1 - 2016 U6 - https://doi.org/10.1021/acssynbio.6b00040 SN - 2161-5063 VL - 5 SP - 710 EP - 720 PB - American Chemical Society CY - Washington ER - TY - GEN A1 - Gorochowski, Thomas E. A1 - Ignatova, Zoya A1 - Bovenberg, Roel A. L. A1 - Roubos, Johannes A. T1 - Trade-offs between tRNA abundance and mRNA secondary structure support smoothing of translation elongation rate T2 - Postprints der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe N2 - Translation of protein from mRNA is a complex multi-step process that occurs at a non-uniform rate. Variability in ribosome speed along an mRNA enables refinement of the proteome and plays a critical role in protein biogenesis. Detailed single protein studies have found both tRNA abundance and mRNA secondary structure as key modulators of translation elongation rate, but recent genome-wide ribosome profiling experiments have not observed significant influence of either on translation efficiency. Here we provide evidence that this results from an inherent trade-off between these factors. We find codons pairing to high-abundance tRNAs are preferentially used in regions of high secondary structure content, while codons read by significantly less abundant tRNAs are located in lowly structured regions. By considering long stretches of high and low mRNA secondary structure in Saccharomyces cerevisiae and Escherichia coli and comparing them to randomized-gene models and experimental expression data, we were able to distinguish clear selective pressures and increased protein expression for specific codon choices. The trade-off between secondary structure and tRNA-concentration based codon choice allows for compensation of their independent effects on translation, helping to smooth overall translational speed and reducing the chance of potentially detrimental points of excessively slow or fast ribosome movement. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 816 KW - Escherichia-coli genome KW - codon adaptation index KW - folding free-energies KW - in-vivo KW - sequence determinants KW - protein-synthesis KW - single ribosomes KW - gene-expression KW - usage KW - bias Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-441340 SN - 1866-8372 IS - 816 ER - TY - JOUR A1 - Gorochowski, Thomas E. A1 - Ignatova, Zoya A1 - Bovenberg, Roel A. L. A1 - Roubos, Johannes A. T1 - Trade-offs between tRNA abundance and mRNA secondary structure support smoothing of translation elongation rate JF - Nucleic acids research N2 - Translation of protein from mRNA is a complex multi-step process that occurs at a non-uniform rate. Variability in ribosome speed along an mRNA enables refinement of the proteome and plays a critical role in protein biogenesis. Detailed single protein studies have found both tRNA abundance and mRNA secondary structure as key modulators of translation elongation rate, but recent genome-wide ribosome profiling experiments have not observed significant influence of either on translation efficiency. Here we provide evidence that this results from an inherent trade-off between these factors. We find codons pairing to high-abundance tRNAs are preferentially used in regions of high secondary structure content, while codons read by significantly less abundant tRNAs are located in lowly structured regions. By considering long stretches of high and low mRNA secondary structure in Saccharomyces cerevisiae and Escherichia coli and comparing them to randomized-gene models and experimental expression data, we were able to distinguish clear selective pressures and increased protein expression for specific codon choices. The trade-off between secondary structure and tRNA-concentration based codon choice allows for compensation of their independent effects on translation, helping to smooth overall translational speed and reducing the chance of potentially detrimental points of excessively slow or fast ribosome movement. Y1 - 2015 U6 - https://doi.org/10.1093/nar/gkv199 SN - 0305-1048 SN - 1362-4962 VL - 43 IS - 6 SP - 3022 EP - 3032 PB - Oxford Univ. Press CY - Oxford ER - TY - JOUR A1 - Hess, Anne-Katrin A1 - Saffert, Paul A1 - Liebeton, Klaus A1 - Ignatova, Zoya T1 - Optimization of Translation Profiles Enhances Protein Expression and Solubility JF - PLoS one N2 - mRNA is translated with a non-uniform speed that actively coordinates co-translational folding of protein domains. Using structure-based homology we identified the structural domains in epoxide hydrolases (EHs) and introduced slow-translating codons to delineate the translation of single domains. These changes in translation speed dramatically improved the solubility of two EHs of metagenomic origin in Escherichia coli. Conversely, the importance of transient attenuation for the folding, and consequently solubility, of EH was evidenced with a member of the EH family from Agrobacterium radiobacter, which partitions in the soluble fraction when expressed in E. coli. Synonymous substitutions of codons shaping the slow-transiting regions to fast-translating codons render this protein insoluble. Furthermore, we show that low protein yield can be enhanced by decreasing the free folding energy of the initial 5'-coding region, which can disrupt mRNA secondary structure and enhance ribosomal loading. This study provides direct experimental evidence that mRNA is not a mere messenger for translation of codons into amino acids but bears an additional layer of information for folding, solubility and expression level of the encoded protein. Furthermore, it provides a general frame on how to modulate and fine-tune gene expression of a target protein. Y1 - 2015 U6 - https://doi.org/10.1371/journal.pone.0127039 SN - 1932-6203 VL - 10 IS - 5 PB - PLoS CY - San Fransisco ER - TY - GEN A1 - Hess, Anne-Katrin A1 - Saffert, Paul A1 - Liebeton, Klaus A1 - Ignatova, Zoya T1 - Optimization of translation profiles enhances protein expression and solubility T2 - Postprints der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe N2 - mRNA is translated with a non-uniform speed that actively coordinates co-translational folding of protein domains. Using structure-based homology we identified the structural domains in epoxide hydrolases (EHs) and introduced slow-translating codons to delineate the translation of single domains. These changes in translation speed dramatically improved the solubility of two EHs of metagenomic origin in Escherichia coli. Conversely, the importance of transient attenuation for the folding, and consequently solubility, of EH was evidenced with a member of the EH family from Agrobacterium radiobacter, which partitions in the soluble fraction when expressed in E. coli. Synonymous substitutions of codons shaping the slow-transiting regions to fast-translating codons render this protein insoluble. Furthermore, we show that low protein yield can be enhanced by decreasing the free folding energy of the initial 5'-coding region, which can disrupt mRNA secondary structure and enhance ribosomal loading. This study provides direct experimental evidence that mRNA is not a mere messenger for translation of codons into amino acids but bears an additional layer of information for folding, solubility and expression level of the encoded protein. Furthermore, it provides a general frame on how to modulate and fine-tune gene expression of a target protein. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 518 KW - transfer-RNA genes KW - codon usage KW - Escherichia coli KW - Epoxide hydrolases KW - messenger-RNA KW - sequence KW - elongation KW - Ribosome KW - mechanism KW - Membrane Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-409574 SN - 1866-8372 IS - 518 ER - TY - JOUR A1 - Hinz, Justyna A1 - Lehnhardt, Lothar A1 - Zakrzewski, Silke A1 - Zhang, Gong A1 - Ignatova, Zoya T1 - Polyglutamine expansion alters the dynamics and molecular architecture of aggregates in dentatorubropallidoluysian atrophy JF - The journal of biological chemistry N2 - Preferential accumulation of mutant proteins in the nucleus has been suggested to be the molecular culprit that confers cellular toxicity in the neurodegenerative disorders caused by polyglutamine (polyQ) expansion. Here, we use dynamic imaging approaches, orthogonal cross-seeding, and composition analysis to examine the dynamics and structure of nuclear and cytoplasmic inclusions of atrophin-1, implicated in dentatorubropallidoluysian atrophy, a polyQ-based disease with complex clinical features. Our results reveal a large heterogeneity in the dynamics of the nuclear inclusions compared with the compact and immobile cytoplasmic aggregates. At least two types of inclusions of expanded atrophin-1 with different mobility of the molecular species and ability to exchange with the surrounding monomer pool coexist in the nucleus. Intriguingly, the enrichment of nuclear inclusions with slow dynamics parallels changes in the aggregate core architecture that are dominated by the polyQ stretch. We propose that the observed complexity in the dynamics of the nuclear inclusions provides a molecular explanation for the enhanced cellular toxicity of the nuclear aggregates in polyQ-based neurodegeneration. Y1 - 2012 U6 - https://doi.org/10.1074/jbc.M111.318915 SN - 0021-9258 VL - 287 IS - 3 SP - 2068 EP - 2078 PB - American Society for Biochemistry and Molecular Biology CY - Bethesda ER - TY - JOUR A1 - Kirchner, Sebastian A1 - Cai, Zhiwei A1 - Rauscher, Robert A1 - Kastelic, Nicolai A1 - Anding, Melanie A1 - Czech, Andreas A1 - Kleizen, Bertrand A1 - Ostedgaard, Lynda S. A1 - Braakman, Ineke A1 - Sheppard, David N. A1 - Ignatova, Zoya T1 - Alteration of protein function by a silent polymorphism linked to tRNA abundance JF - PLoS biology N2 - Synonymous single nucleotide polymorphisms (sSNPs) are considered neutral for protein function, as by definition they exchange only codons, not amino acids. We identified an sSNP that modifies the local translation speed of the cystic fibrosis transmembrane conductance regulator (CFTR), leading to detrimental changes to protein stability and function. This sSNP introduces a codon pairing to a low-abundance tRNA that is particularly rare in human bronchial epithelia, but not in other human tissues, suggesting tissue-specific effects of this sSNP. Up-regulation of the tRNA cognate to the mutated codon counteracts the effects of the sSNP and rescues protein conformation and function. Our results highlight the wide-ranging impact of sSNPs, which invert the programmed local speed of mRNA translation and provide direct evidence for the central role of cellular tRNA levels in mediating the actions of sSNPs in a tissue-specific manner. Y1 - 2017 U6 - https://doi.org/10.1371/journal.pbio.2000779 SN - 1545-7885 VL - 15 PB - PLoS CY - San Fransisco ER - TY - JOUR A1 - Kirchner, Sebastian A1 - Ignatova, Zoya T1 - Emerging roles of tRNA in adaptive translation, signalling dynamics and disease JF - Nature reviews N2 - tRNAs, nexus molecules between mRNAs and proteins, have a central role in translation. Recent discoveries have revealed unprecedented complexity of tRNA biosynthesis, modification patterns, regulation and function. In this Review, we present emerging concepts regarding how tRNA abundance is dynamically regulated and how tRNAs (and their nucleolytic fragments) are centrally involved in stress signalling and adaptive translation, operating across a wide range of timescales. Mutations in tRNAs or in genes affecting tRNA biogenesis are also linked to complex human diseases with surprising heterogeneity in tissue vulnerability, and we highlight cell-specific aspects that modulate the disease penetrance of tRNA-based pathologies. Y1 - 2015 U6 - https://doi.org/10.1038/nrg3861 SN - 1471-0056 SN - 1471-0064 VL - 16 IS - 2 SP - 98 EP - 112 PB - Nature Publ. Group CY - London ER - TY - GEN A1 - Lukoszek, Radoslaw A1 - Feist, Peter A1 - Ignatova, Zoya T1 - Insights into the adaptive response of Arabidopsis thaliana to prolonged thermal stress by ribosomal profiling and RNA-Seq T2 - BMC plant biology N2 - Background: Environmental stress puts organisms at risk and requires specific stress-tailored responses to maximize survival. Long-term exposure to stress necessitates a global reprogramming of the cellular activities at different levels of gene expression. Results: Here, we use ribosome profiling and RNA sequencing to globally profile the adaptive response of Arabidopsis thaliana to prolonged heat stress. To adapt to long heat exposure, the expression of many genes is modulated in a coordinated manner at a transcriptional and translational level. However, a significant group of genes opposes this trend and shows mainly translational regulation. Different secondary structure elements are likely candidates to play a role in regulating translation of those genes. Conclusions: Our data also uncover on how the subunit stoichiometry of multimeric protein complexes in plastids is maintained upon heat exposure. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 438 KW - translation KW - ribosome profiling KW - transcription KW - RNA-Seq KW - secondary structure KW - G-quadruplexes, KW - heat stress response Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-407262 ER - TY - JOUR A1 - Lukoszek, Radoslaw A1 - Feist, Peter A1 - Ignatova, Zoya T1 - Insights into the adaptive response of Arabidopsis thaliana to prolonged thermal stress by ribosomal profiling and RNA-Seq JF - BMC plant biology N2 - Background: Environmental stress puts organisms at risk and requires specific stress-tailored responses to maximize survival. Long-term exposure to stress necessitates a global reprogramming of the cellular activities at different levels of gene expression. Results: Here, we use ribosome profiling and RNA sequencing to globally profile the adaptive response of Arabidopsis thaliana to prolonged heat stress. To adapt to long heat exposure, the expression of many genes is modulated in a coordinated manner at a transcriptional and translational level. However, a significant group of genes opposes this trend and shows mainly translational regulation. Different secondary structure elements are likely candidates to play a role in regulating translation of those genes. Conclusions: Our data also uncover on how the subunit stoichiometry of multimeric protein complexes in plastids is maintained upon heat exposure. KW - Translation KW - Ribosome profiling KW - Transcription KW - RNA-Seq KW - Secondary structure KW - G-quadruplexes KW - Heat stress response Y1 - 2016 U6 - https://doi.org/10.1186/s12870-016-0915-0 SN - 1471-2229 VL - 16 PB - BioMed Central CY - London ER - TY - JOUR A1 - Lukoszek, Radoslaw A1 - Müller-Röber, Bernd A1 - Ignatova, Zoya T1 - Interplay between polymerase II- and polymerase III-assisted expression of overlapping genes JF - FEBS letters : the journal for rapid publication of short reports in molecular biosciences N2 - Up to 15% of the genes in different genomes overlap. This architecture, although beneficial for the genome size, represents an obstacle for simultaneous transcription of both genes. Here we analyze the interference between RNA-polymerase II (Pol II) and RNA-polymerase III (Pol III) when transcribing their target genes encoded on opposing strands within the same DNA fragment in Arabidopsis thaliana. The expression of a Pol II-dependent protein-coding gene negatively correlated with the transcription of a Pol III-dependent, tRNA-coding gene set. We suggest that the architecture of the overlapping genes introduces an additional layer of control of gene expression. (C) 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. KW - Gene expression KW - Transcription KW - tRNA KW - Nested and overlapping genes KW - Arabidopsis thaliana Y1 - 2013 U6 - https://doi.org/10.1016/j.febslet.2013.09.033 SN - 0014-5793 SN - 1873-3468 VL - 587 IS - 22 SP - 3692 EP - 3695 PB - Elsevier CY - Amsterdam ER - TY - JOUR A1 - Miettinen, Markus S. A1 - Knecht, Volker A1 - Monticelli, Luca A1 - Ignatova, Zoya T1 - Assessing polyglutamine conformation in the nucleating event by molecular dynamics simulations JF - The journal of physical chemistry : B, Condensed matter, materials, surfaces, interfaces & biophysical chemistry N2 - Polyglutamine (polyQ) diseases comprise a group of dominantly inherited pathology caused by an expansion of an unstable polyQ stretch which is presumed to form beta-sheets. Similar to other amyloid pathologies, polyQ amyloidogenesis occurs via a nucleated polymerization mechanism, and proceeds through energetically unfavorable nucleus whose existence and structure are difficult to detect. Here, we use atomistic molecular dynamics simulations in explicit solvent to assess the conformation of the polyQ stretch in the nucleus that initiates polyQ fibrillization. Comparison of the kinetic stability of various structures of polyQ peptide with a Q-length in the pathological range (Q(40)) revealed that steric zipper or nanotube-like structures (beta-nanotube or beta-pseudohelix) are not kinetically stable enough to serve as a template to initiate polyQ fibrillization as opposed to beta-hairpin-based (beta-sheet and beta-sheetstack) or alpha-helical conformations. The selection of different structures of the polyQ stretch in the aggregation-initiating event may provide an alternative explanation for polyQ aggregate polymorphism. Y1 - 2012 U6 - https://doi.org/10.1021/jp305065c SN - 1520-6106 VL - 116 IS - 34 SP - 10259 EP - 10265 PB - American Chemical Society CY - Washington ER - TY - JOUR A1 - Miettinen, Markus S. A1 - Monticelli, Luca A1 - Nedumpully-Govindan, Praveen A1 - Knecht, Volker A1 - Ignatova, Zoya T1 - Stable polyglutamine dimers can contain beta-hairpins with interdigitated side chains but not a-helices, alpha-nanotubes, beta-pseudohelices, or steric zippers JF - Biophysical journal N2 - A common thread connecting nine fatal neurodegenerative protein aggregation diseases is an abnormally expanded polyglutamine tract found in the respective proteins. Although the structure of this tract in the large mature aggregates is increasingly well described, its structure in the small early aggregates remains largely unknown. As experimental evidence suggests that the most toxic species along the aggregation pathway are the small early ones, developing strategies to alleviate disease pathology calls for understanding the structure of polyglutamine peptides in the early stages of aggregation. Here, we present a criterion, grounded in available experimental data, that allows for using kinetic stability of dimers to assess whether a given polyglutamine conformer can be on the aggregation path. We then demonstrate that this criterion can be assessed using present-day molecular dynamics simulations. We find that although the a-helical conformer of polyglutamine is very stable, dimers of a-helices lack the kinetic stability necessary to support further oligomerization. Dimers of steric zipper, beta-nanotube, and beta-pseudohelix conformers are also too short-lived to initiate aggregation. The beta-hairpin-containing conformers, instead, invariably form very stable dimers when their side chains are interdigitated. Combining these findings with the implications of recent solid-state NMR data on mature fibrils, we propose a possible pathway for the initial stages of polyglutamine aggregation, in which beta-hairpin-containing conformers act as templates for fibril formation. Y1 - 2014 U6 - https://doi.org/10.1016/j.bpj.2014.02.027 SN - 0006-3495 SN - 1542-0086 VL - 106 IS - 8 SP - 1721 EP - 1728 PB - Cell Press CY - Cambridge ER - TY - CHAP A1 - Miettinen, Markus S. A1 - Monticelli, Luca A1 - Nedumpully-Govindan, Praveen A1 - Knecht, Volker A1 - Ignatova, Zoya T1 - Initiating polyglutamine aggregation - computational clarification of the structural details T2 - Biophysical journal Y1 - 2015 SN - 0006-3495 SN - 1542-0086 VL - 108 IS - 2 SP - 386A EP - 386A PB - Cell Press CY - Cambridge ER - TY - JOUR A1 - Puri, Pranav A1 - Wetzel, Collin A1 - Saffert, Paul A1 - Gaston, Kirk W. A1 - Russell, Susan P. A1 - Varela, Juan A. Cordero A1 - van der Vlies, Pieter A1 - Zhang, Gong A1 - Limbach, Patrick A. A1 - Ignatova, Zoya A1 - Poolman, Bert T1 - Systematic identification of tRNAome and its dynamics in Lactococcus lactis JF - Molecular microbiology N2 - Transfer RNAs (tRNAs) through their abundance and modification pattern significantly influence protein translation. Here, we present a systematic analysis of the tRNAome of Lactococcus lactis. Using the next-generation sequencing approach, we identified 40 tRNAs which carry 16 different post-transcriptional modifications as revealed by mass spectrometry analysis. While small modifications are located in the tRNA body, hypermodified nucleotides are mainly present in the anticodon loop, which through wobbling expand the decoding potential of the tRNAs. Using tRNA-based microarrays, we also determined the dynamics in tRNA abundance upon changes in the growth rate and heterologous protein overexpression stress. With a fourfold increase in the growth rate, the relative abundance of tRNAs cognate to low abundance codons decrease, while the tRNAs cognate to major codons remain mostly unchanged. Significant changes in the tRNA abundances are observed upon protein overexpression stress, which does not correlate with the codon usage of the overexpressed gene but rather reflects the altered expression of housekeeping genes. Y1 - 2014 U6 - https://doi.org/10.1111/mmi.12710 SN - 0950-382X SN - 1365-2958 VL - 93 IS - 5 SP - 944 EP - 956 PB - Wiley-Blackwell CY - Hoboken ER - TY - JOUR A1 - Roethlein, Christoph A1 - Miettinen, Markus S. A1 - Borwankar, Tejas A1 - Buerger, Joerg A1 - Mielke, Thorsten A1 - Kumke, Michael Uwe A1 - Ignatova, Zoya T1 - Architecture of polyglutamine-containing fibrils from time-resolved fluorescence decay JF - The journal of biological chemistry N2 - The disease risk and age of onset of Huntington disease (HD) and nine other repeat disorders strongly depend on the expansion of CAG repeats encoding consecutive polyglutamines (polyQ) in the corresponding disease protein. PolyQ length-dependent misfolding and aggregation are the hallmarks of CAG pathologies. Despite intense effort, the overall structure of these aggregates remains poorly understood. Here, we used sensitive time-dependent fluorescent decay measurements to assess the architecture of mature fibrils of huntingtin (Htt) exon 1 implicated in HD pathology. Varying the position of the fluorescent labels in the Htt monomer with expanded 51Q (Htt51Q) and using structural models of putative fibril structures, we generated distance distributions between donors and acceptors covering all possible distances between the monomers or monomer dimensions within the polyQ amyloid fibril. Using Monte Carlo simulations, we systematically scanned all possible monomer conformations that fit the experimentally measured decay times. Monomers with four-stranded 51Q stretches organized into five-layered beta-sheets with alternating N termini of the monomers perpendicular to the fibril axis gave the best fit to our data. Alternatively, the core structure of the polyQ fibrils might also be a zipper layer with antiparallel four-stranded stretches as this structure showed the next best fit. All other remaining arrangements are clearly excluded by the data. Furthermore, the assessed dimensions of the polyQ stretch of each monomer provide structural evidence for the observed polyQ length threshold in HD pathology. Our approach can be used to validate the effect of pharmacological substances that inhibit or alter amyloid growth and structure. Y1 - 2014 U6 - https://doi.org/10.1074/jbc.M114.581991 SN - 0021-9258 SN - 1083-351X VL - 289 IS - 39 SP - 26817 EP - 26828 PB - American Society for Biochemistry and Molecular Biology CY - Bethesda ER - TY - GEN A1 - Roethlein, Christoph A1 - Miettinen, Markus S. A1 - Ignatova, Zoya T1 - A flexible approach to assess fluorescence decay functions in complex energy transfer systems T2 - Postprints der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe 819 N2 - Background: Time-correlated Forster resonance energy transfer (FRET) probes molecular distances with greater accuracy than intensity-based calculation of FRET efficiency and provides a powerful tool to study biomolecular structure and dynamics. Moreover, time-correlated photon count measurements bear additional information on the variety of donor surroundings allowing more detailed differentiation between distinct structural geometries which are typically inaccessible to general fitting solutions. Results: Here we develop a new approach based on Monte Carlo simulations of time-correlated FRET events to estimate the time-correlated single photon counts (TCSPC) histograms in complex systems. This simulation solution assesses the full statistics of time-correlated photon counts and distance distributions of fluorescently labeled biomolecules. The simulations are consistent with the theoretical predictions of the dye behavior in FRET systems with defined dye distances and measurements of randomly distributed dye solutions. We validate the simulation results using a highly heterogeneous aggregation system and explore the conditions to use this tool in complex systems. Conclusion: This approach is powerful in distinguishing distance distributions in a wide variety of experimental setups, thus providing a versatile tool to accurately distinguish between different structural assemblies in highly complex systems. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 819 KW - time resolved FRET KW - Monte-Carlo simulations KW - complex heterogeneous systems KW - protein aggregation Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-427557 SN - 1866-8372 IS - 819 ER - TY - JOUR A1 - Röthlein, Christoph A1 - Miettinen, Markus S. A1 - Ignatova, Zoya T1 - A flexible approach to assess fluorescence decay functions in complex energy transfer systems JF - BMC biophysics N2 - Background: Time-correlated Forster resonance energy transfer (FRET) probes molecular distances with greater accuracy than intensity-based calculation of FRET efficiency and provides a powerful tool to study biomolecular structure and dynamics. Moreover, time-correlated photon count measurements bear additional information on the variety of donor surroundings allowing more detailed differentiation between distinct structural geometries which are typically inaccessible to general fitting solutions. Results: Here we develop a new approach based on Monte Carlo simulations of time-correlated FRET events to estimate the time-correlated single photon counts (TCSPC) histograms in complex systems. This simulation solution assesses the full statistics of time-correlated photon counts and distance distributions of fluorescently labeled biomolecules. The simulations are consistent with the theoretical predictions of the dye behavior in FRET systems with defined dye distances and measurements of randomly distributed dye solutions. We validate the simulation results using a highly heterogeneous aggregation system and explore the conditions to use this tool in complex systems. Conclusion: This approach is powerful in distinguishing distance distributions in a wide variety of experimental setups, thus providing a versatile tool to accurately distinguish between different structural assemblies in highly complex systems. KW - Time resolved FRET KW - Monte-Carlo simulations KW - Complex heterogeneous systems KW - Protein aggregation Y1 - 2015 U6 - https://doi.org/10.1186/s13628-015-0020-z SN - 2046-1682 VL - 8 PB - BioMed Central CY - London ER - TY - JOUR A1 - Saffert, Paul A1 - Adamla, Frauke A1 - Schieweck, Rico A1 - Atkins, John F. A1 - Ignatova, Zoya T1 - An Expanded CAG Repeat in Huntingtin Causes+1 Frameshifting JF - The journal of biological chemistry N2 - Maintenance of triplet decoding is crucial for the expression of functional protein because deviations either into the -1 or +1 reading frames are often non-functional. We report here that expression of huntingtin (Htt) exon 1 with expanded CAG repeats, implicated in Huntington pathology, undergoes a sporadic +1 frameshift to generate from the CAG repeat a trans-frame AGC repeat-encoded product. This +1 recoding is exclusively detected in pathological Htt variants, i.e. those with expanded repeats with more than 35 consecutive CAG codons. An atypical +1 shift site, UUC C at the 5 end of CAG repeats, which has some resemblance to the influenza A virus shift site, triggers the +1 frameshifting and is enhanced by the increased propensity of the expanded CAG repeats to form a stem-loop structure. The +1 trans-frame-encoded product can directly influence the aggregation of the parental Htt exon 1. KW - aggregation KW - Huntington disease KW - translation KW - translation regulation KW - trinucleotide repeat disease KW - frameshifting KW - seeding Y1 - 2016 U6 - https://doi.org/10.1074/jbc.M116.744326 SN - 0021-9258 SN - 1083-351X VL - 291 SP - 18505 EP - 18513 PB - American Society for Biochemistry and Molecular Biology CY - Bethesda ER - TY - JOUR A1 - Valleriani, Angelo A1 - Ignatova, Zoya A1 - Nagar, Apoorva A1 - Lipowsky, Reinhard T1 - Turnover of messenger RNA : polysome statistics beyond the steady state N2 - The interplay between turnover or degradation and ribosome loading of messenger RNA (mRNA) is studied theoretically using a stochastic model that is motivated by recent experimental results. Random mRNA degradation affects the statistics of polysomes, i.e., the statistics of the number of ribosomes per mRNA as extracted from cells. Since ribosome loading of newly created mRNA chains requires some time to reach steady state, a fraction of the extracted mRNA/ ribosome complexes does not represent steady state conditions. As a consequence, the mean ribosome density obtained from the extracted complexes is found to be inversely proportional to the mRNA length. On the other hand, the ribosome density profile shows an exponential decrease along the mRNA for prokaryotes and becomes uniform in eukaryotic cells. Copyright (C) EPLA, 2010 Y1 - 2010 UR - http://iopscience.iop.org/0295-5075/ U6 - https://doi.org/10.1209/0295-5075/89/58003 SN - 0295-5075 ER - TY - JOUR A1 - Valleriani, Angelo A1 - Zhang, Gong A1 - Nagar, Apoorva A1 - Ignatova, Zoya A1 - Lipowsky, Reinhard T1 - Length-dependent translation of messenger RNA by ribosomes JF - Physical review : E, Statistical, nonlinear and soft matter physics N2 - A simple measure for the efficiency of protein synthesis by ribosomes is provided by the steady state amount of protein per messenger RNA (mRNA), the so-called translational ratio, which is proportional to the translation rate. Taking the degradation of mRNA into account, we show theoretically that both the translation rate and the translational ratio decrease with increasing mRNA length, in agreement with available experimental data for the prokaryote Escherichia coli. We also show that, compared to prokaryotes, mRNA degradation in eukaryotes leads to a less rapid decrease of the translational ratio. This finding is consistent with the fact that, compared to prokaryotes, eukaryotes tend to have longer proteins. Y1 - 2011 U6 - https://doi.org/10.1103/PhysRevE.83.042903 SN - 1539-3755 VL - 83 IS - 4 PB - American Physical Society CY - College Park ER - TY - JOUR A1 - Varshney, Nishant Kumar A1 - Kumar, R. Suresh A1 - Ignatova, Zoya A1 - Prabhune, Asmita A1 - Pundle, Archana A1 - Dodson, Eleanor A1 - Suresh, C. G. T1 - Crystallization and X-ray structure analysis of a thermostable penicillin G acylase from Alcaligenes faecalis JF - Acta crystallographica : Section F, Structural biology communications N2 - The enzyme penicillin G acylase (EC 3.5.1.11) catalyzes amide-bond cleavage in benzylpenicillin (penicillin G) to yield 6-aminopenicillanic acid, an intermediate chemical used in the production of semisynthetic penicillins. A thermostable penicillin G acylase from Alcaligenes faecalis (AfPGA) has been crystallized using the hanging-drop vapour-diffusion method in two different space groups: C2221, with unit-cell parameters a = 72.9, b = 86.0, c = 260.2 angstrom, and P41212, with unit-cell parameters a = b = 85.6, c = 298.8 angstrom. Data were collected at 293 K and the structure was determined using the molecular-replacement method. Like other penicillin acylases, AfPGA belongs to the N-terminal nucleophilic hydrolase superfamily, has undergone post-translational processing and has a serine as the N-terminal residue of the beta-chain. A disulfide bridge has been identified in the structure that was not found in the other two known penicillin G acylase structures. The presence of the disulfide bridge is perceived to be one factor that confers higher stability to this enzyme. KW - thermostability KW - disulfide bridges KW - Ntn hydrolases KW - orthorhombic form KW - tetragonal form KW - calcium binding Y1 - 2012 U6 - https://doi.org/10.1107/S1744309111053930 SN - 1744-3091 VL - 68 IS - 3 SP - 273 EP - 277 PB - Wiley-Blackwell CY - Malden ER - TY - JOUR A1 - Weissenborn, Christine A1 - Ignatov, Tanja A1 - Ochel, Hans-Joachim A1 - Costa, Serban Dan A1 - Zenclussen, Ana Claudia A1 - Ignatova, Zoya A1 - Ignatov, Atanas T1 - GPER functions as a tumor suppressor in triple-negative breast cancer cells JF - Journal of cancer research and clinical oncology : official organ of the Deutsche Krebsgesellschaft N2 - We investigated the role of GPER as a potential tumor suppressor in triple-negative breast cancer cells MDA-MB-231 and MDA-MB-468 using cell cycle analysis and apoptosis assay. The constitutive activity of GPER was investigated. GPER-specific activation with G-1 agonist inhibited breast cancer cell growth in concentration-dependent manner via induction of the cell cycle arrest in G2/M phase, enhanced phosphorylation of histone H3 and caspase-3-mediated apoptosis. Analysis of the methylation status of the GPER promoter in the triple-negative breast cancer cells and in tissues derived from breast cancer patients revealed that GPER amount is regulated by epigenetic mechanisms and GPER expression is inactivated by promoter methylation. Furthermore, GPER expression was induced by stress factors, such as radiation, and GPER amount inversely correlated with the p53 expression level. Overall, our results establish the protective role in breast cancer tumorigenesis, and the cell surface expression of GPER makes it an excellent potential therapeutic target for triple-negative breast cancer. KW - GPER KW - GPR30 KW - Breast cancer KW - Tumor suppression KW - TNBC Y1 - 2014 U6 - https://doi.org/10.1007/s00432-014-1620-8 SN - 0171-5216 SN - 1432-1335 VL - 140 IS - 5 SP - 713 EP - 723 PB - Springer CY - New York ER - TY - JOUR A1 - Yuryev, Ruslan A1 - Kasche, Volker A1 - Ignatova, Zoya A1 - Galunsky, Boris T1 - Improved A. faecalis penicillin amidase mutant retains the thermodynamic and pH stability of the wild type enzyme N2 - Penicillin amidase from Alacaligenes faecalis is an attractive biocatalyst for hydrolysis of penicillin G for production of 6-aminopenicillanic acid, which is used in the synthesis of semi-synthetic beta-lactam antibiotics. Recently a mutant of this enzyme with extended C-terminus of the A-chain comprising parts of the connecting linker peptide was constructed. Its turnover number for the hydrolysis of penicillin G was 140 s(-1), about twice of the value for the wild-type enzyme (80 s(-1)). At the same time the specificity constant was improved about three-fold. The wild- type and the mutant enzymes showed similar pH stability suggesting that the linker peptide fragment covalently attached to the A-chain does not alter the electrostatic interactions in the protein core. Although the global stability of A. faecalis wild-type enzyme and the T206GS213G variant does not differ, the presence of the linker fragment stabilizes the domains interface, as evidenced by the monophasic transition of the mutant enzyme from folded to unfolded state during urea-induced denaturation. The high stability and activity of the mutant enzyme provides a rationale to use it as a biocatalyst in the industrial processes, where the enzyme must be more robust to fluctuations in the operational conditions. Y1 - 2010 UR - http://springerlink.metapress.com/content/1573-4943/ U6 - https://doi.org/10.1007/s10930-010-9238-4 SN - 1572-3887 ER - TY - JOUR A1 - Zhang, Gong A1 - Fedyunin, Ivan A1 - Kirchner, Sebastian A1 - Xiao, Chuanle A1 - Valleriani, Angelo A1 - Ignatova, Zoya T1 - FANSe: an accurate algorithm for quantitative mapping of large scale sequencing reads JF - Nucleic acids research N2 - The most crucial step in data processing from high-throughput sequencing applications is the accurate and sensitive alignment of the sequencing reads to reference genomes or transcriptomes. The accurate detection of insertions and deletions (indels) and errors introduced by the sequencing platform or by misreading of modified nucleotides is essential for the quantitative processing of the RNA-based sequencing (RNA-Seq) datasets and for the identification of genetic variations and modification patterns. We developed a new, fast and accurate algorithm for nucleic acid sequence analysis, FANSe, with adjustable mismatch allowance settings and ability to handle indels to accurately and quantitatively map millions of reads to small or large reference genomes. It is a seed-based algorithm which uses the whole read information for mapping and high sensitivity and low ambiguity are achieved by using short and non-overlapping reads. Furthermore, FANSe uses hotspot score to prioritize the processing of highly possible matches and implements modified Smith-Watermann refinement with reduced scoring matrix to accelerate the calculation without compromising its sensitivity. The FANSe algorithm stably processes datasets from various sequencing platforms, masked or unmasked and small or large genomes. It shows a remarkable coverage of low-abundance mRNAs which is important for quantitative processing of RNA-Seq datasets. Y1 - 2012 U6 - https://doi.org/10.1093/nar/gks196 SN - 0305-1048 VL - 40 IS - 11 PB - Oxford Univ. Press CY - Oxford ER - TY - JOUR A1 - Zhang, Gong A1 - Fedyunin, Ivan A1 - Miekley, Oskar A1 - Valleriani, Angelo A1 - Moura, Alessandro A1 - Ignatova, Zoya T1 - Global and local depletion of ternary complex limits translational elongation N2 - The translation of genetic information according to the sequence of the mRNA template occurs with high accuracy and fidelity. Critical events in each single step of translation are selection of transfer RNA (tRNA), codon reading and tRNA-regeneration for a new cycle. We developed a model that accurately describes the dynamics of single elongation steps, thus providing a systematic insight into the sensitivity of the mRNA translation rate to dynamic environmental conditions. Alterations in the concentration of the aminoacylated tRNA can transiently stall the ribosomes during translation which results, as suggested by the model, in two outcomes: either stress-induced change in the tRNA availability triggers the premature termination of the translation and ribosomal dissociation, or extensive demand for one tRNA species results in a competition between frameshift to an aberrant open-reading frame and ribosomal drop-off. Using the bacterial Escherichia coli system, we experimentally draw parallels between these two possible mechanisms. Y1 - 2010 UR - http://nar.oxfordjournals.org/ U6 - https://doi.org/10.1093/Nar/Gkq196 SN - 0305-1048 ER - TY - JOUR A1 - Zhang, Gong A1 - Hubalewska, Magdalena A1 - Ignatova, Zoya T1 - Transient ribosomal attenuation coordinates protein synthesis and co-translational folding N2 - Clustered codons that pair to low-abundance tRNA isoacceptors can form slow-translating regions in the mRNA and cause transient ribosomal arrest. We report that folding efficiency of the Escherichia coli multidomain protein Sufl can be severely perturbed by alterations in ribosome-mediated translational attenuation. Such alterations were achieved by global acceleration of the translation rate with tRNA excess in vitro or by synonymous substitutions to codons with highly abundant tRNAs both in vitro and in vivo. Conversely, the global slow-down of the translation rate modulated by low temperature suppresses the deleterious effect of the altered translational attenuation pattern. We propose that local discontinuous translation temporally separates the translation of segments of the peptide chain and actively coordinates their co-translational folding. Y1 - 2009 UR - http://www.nature.com/nsmb/ U6 - https://doi.org/10.1038/Nsmb.1554 SN - 1545-9985 ER - TY - GEN A1 - Zhang, Gong A1 - Ignatova, Zoya T1 - Generic algorithm to predict the speed of translational elongation : implications for protein biogenesis N2 - Synonymous codon usage and variations in the level of isoaccepting tRNAs exert a powerful selective force on translation fidelity. We have developed an algorithm to evaluate the relative rate of translation which allows large-scale comparisons of the non-uniform translation rate on the protein biogenesis. Using the complete genomes of Escherichia coli and Bacillus subtilis we show that stretches of codons pairing to minor tRNAs form putative sites to locally attenuate translation; thereby the tendency is to cluster in near proximity whereas long contiguous stretches of slow-translating triplets are avoided. The presence of slow-translating segments positively correlates with the protein length irrespective of the protein abundance. The slow-translating clusters are predominantly located down-stream of the domain boundaries presumably to fine-tune translational accuracy with the folding fidelity of multidomain proteins. Translation attenuation patterns at highly structurally and functionally conserved domains are preserved across the species suggesting a concerted selective pressure on the codon selection and species-specific tRNA abundance in these regions. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - paper 132 Y1 - 2009 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus-45007 ER - TY - JOUR A1 - Zhang, Gong A1 - Ignatova, Zoya T1 - Folding at the birth of the nascent chain: coordinating translation with co-translational folding JF - Current opinion in structural biology : review of all advances ; evaluation of key references ; comprehensive listing of papers N2 - In the living cells, the folding of many proteins is largely believed to begin co-translationally, during their biosynthesis at the ribosomes. In the ribosomal tunnel, the nascent peptide may establish local interactions and stabilize alpha-helical structures. Long-range contacts are more likely outside the ribosomes after release of larger segments of the nascent chain. Examples suggest that domains can attain native-like structure on the ribosome with and without population of folding intermediates. The co-translational folding is limited by the speed of the gradual extrusion of the nascent peptide which imposes conformational restraints on its folding landscape. Recent experimental and in silico modeling studies indicate that translation kinetics fine-tunes co-translational folding by providing a time delay for sequential folding of distinct portions of the nascent chain. Y1 - 2011 U6 - https://doi.org/10.1016/j.sbi.2010.10.008 SN - 0959-440X VL - 21 IS - 1 SP - 25 EP - 31 PB - Elsevier CY - London ER - TY - JOUR A1 - Zhang, Gong A1 - Lukoszek, Radoslaw A1 - Müller-Röber, Bernd A1 - Ignatova, Zoya T1 - Different sequence signatures in the upstream regions of plant and animal tRNA genes shape distinct modes of regulation JF - Nucleic acids research N2 - In eukaryotes, the transcription of tRNA genes is initiated by the concerted action of transcription factors IIIC (TFIIIC) and IIIB (TFIIIB) which direct the recruitment of polymerase III. While TFIIIC recognizes highly conserved, intragenic promoter elements, TFIIIB binds to the non-coding 5'-upstream regions of the tRNA genes. Using a systematic bioinformatic analysis of 11 multicellular eukaryotic genomes we identified a highly conserved TATA motif followed by a CAA-motif in the tRNA upstream regions of all plant genomes. Strikingly, the 5'-flanking tRNA regions of the animal genomes are highly heterogeneous and lack a common conserved sequence signature. Interestingly, in the animal genomes the tRNA species that read the same codon share conserved motifs in their upstream regions. Deep-sequencing analysis of 16 human tissues revealed multiple splicing variants of two of the TFIIIB subunits, Bdp1 and Brf1, with tissue-specific expression patterns. These multiple forms most likely modulate the TFIIIB-DNA interactions and explain the lack of a uniform signature motif in the tRNA upstream regions of animal genomes. The anticodon-dependent 5'-flanking motifs provide a possible mechanism for independent regulation of the tRNA transcription in various human tissues. Y1 - 2011 U6 - https://doi.org/10.1093/nar/gkq1257 SN - 0305-1048 VL - 39 IS - 8 SP - 3331 EP - 3339 PB - Oxford Univ. Press CY - Oxford ER -