@article{KirchnerCaiRauscheretal.2017, author = {Kirchner, Sebastian and Cai, Zhiwei and Rauscher, Robert and Kastelic, Nicolai and Anding, Melanie and Czech, Andreas and Kleizen, Bertrand and Ostedgaard, Lynda S. and Braakman, Ineke and Sheppard, David N. and Ignatova, Zoya}, title = {Alteration of protein function by a silent polymorphism linked to tRNA abundance}, series = {PLoS biology}, volume = {15}, journal = {PLoS biology}, publisher = {PLoS}, address = {San Fransisco}, issn = {1545-7885}, doi = {10.1371/journal.pbio.2000779}, pages = {29}, year = {2017}, abstract = {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.}, language = {en} } @article{LukoszekFeistIgnatova2016, author = {Lukoszek, Radoslaw and Feist, Peter and Ignatova, Zoya}, title = {Insights into the adaptive response of Arabidopsis thaliana to prolonged thermal stress by ribosomal profiling and RNA-Seq}, series = {BMC plant biology}, volume = {16}, journal = {BMC plant biology}, publisher = {BioMed Central}, address = {London}, issn = {1471-2229}, doi = {10.1186/s12870-016-0915-0}, pages = {13}, year = {2016}, abstract = {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.}, language = {en} } @article{SaffertAdamlaSchiewecketal.2016, author = {Saffert, Paul and Adamla, Frauke and Schieweck, Rico and Atkins, John F. and Ignatova, Zoya}, title = {An Expanded CAG Repeat in Huntingtin Causes+1 Frameshifting}, series = {The journal of biological chemistry}, volume = {291}, journal = {The journal of biological chemistry}, publisher = {American Society for Biochemistry and Molecular Biology}, address = {Bethesda}, issn = {0021-9258}, doi = {10.1074/jbc.M116.744326}, pages = {18505 -- 18513}, year = {2016}, abstract = {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.}, language = {en} } @article{GorochowskiAycilarKucukgozeBovenbergetal.2016, author = {Gorochowski, Thomas E. and Aycilar-Kucukgoze, Irem and Bovenberg, Roel A. L. and Roubos, Johannes A. and Ignatova, Zoya}, title = {A Minimal Model of Ribosome Allocation Dynamics Captures Trade-offs in Expression between Endogenous and Synthetic Genes}, series = {ACS synthetic biology}, volume = {5}, journal = {ACS synthetic biology}, publisher = {American Chemical Society}, address = {Washington}, issn = {2161-5063}, doi = {10.1021/acssynbio.6b00040}, pages = {710 -- 720}, year = {2016}, abstract = {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.}, language = {en} } @article{AvcilarKucukgozeBartholomaeusVarelaetal.2016, author = {Avcilar-Kucukgoze, Irem and Bartholom{\"a}us, Alexander and Varela, Juan A. Cordero and Kaml, Robert Franz-Xaver and Neubauer, Peter and Budisa, Nediljko and Ignatova, Zoya}, title = {Discharging tRNAs: a tug of war between translation and detoxification in Escherichia coli}, series = {Nucleic acids research}, volume = {44}, journal = {Nucleic acids research}, publisher = {Oxford Univ. Press}, address = {Oxford}, issn = {0305-1048}, doi = {10.1093/nar/gkw697}, pages = {8324 -- 8334}, year = {2016}, abstract = {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.}, language = {en} } @article{BartholomaeusFedyuninFeistetal.2016, author = {Bartholom{\"a}us, Alexander and Fedyunin, Ivan and Feist, Peter and Sin, Celine and Zhang, Gong and Valleriani, Angelo and Ignatova, Zoya}, title = {Bacteria differently regulate mRNA abundance to specifically respond to various stresses}, series = {Geology}, volume = {374}, journal = {Geology}, publisher = {Royal Society}, address = {London}, issn = {1364-503X}, doi = {10.1098/rsta.2015.0069}, pages = {16}, year = {2016}, abstract = {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.}, language = {en} } @article{RoethleinMiettinenIgnatova2015, author = {R{\"o}thlein, Christoph and Miettinen, Markus S. and Ignatova, Zoya}, title = {A flexible approach to assess fluorescence decay functions in complex energy transfer systems}, series = {BMC biophysics}, volume = {8}, journal = {BMC biophysics}, publisher = {BioMed Central}, address = {London}, issn = {2046-1682}, doi = {10.1186/s13628-015-0020-z}, pages = {10}, year = {2015}, abstract = {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.}, language = {en} } @article{AdamlaIgnatova2015, author = {Adamla, Frauke and Ignatova, Zoya}, title = {Somatic expression of unc-54 and vha-6 mRNAs declines but not pan-neuronal rgef-1 and unc-119 expression in aging Caenorhabditis elegans}, series = {Scientific reports}, volume = {5}, journal = {Scientific reports}, publisher = {Nature Publ. Group}, address = {London}, issn = {2045-2322}, doi = {10.1038/srep10692}, pages = {10}, year = {2015}, abstract = {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.}, language = {en} } @article{HessSaffertLiebetonetal.2015, author = {Hess, Anne-Katrin and Saffert, Paul and Liebeton, Klaus and Ignatova, Zoya}, title = {Optimization of Translation Profiles Enhances Protein Expression and Solubility}, series = {PLoS one}, volume = {10}, journal = {PLoS one}, number = {5}, publisher = {PLoS}, address = {San Fransisco}, issn = {1932-6203}, doi = {10.1371/journal.pone.0127039}, pages = {14}, year = {2015}, abstract = {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.}, language = {en} } @article{GorochowskiIgnatovaBovenbergetal.2015, author = {Gorochowski, Thomas E. and Ignatova, Zoya and Bovenberg, Roel A. L. and Roubos, Johannes A.}, title = {Trade-offs between tRNA abundance and mRNA secondary structure support smoothing of translation elongation rate}, series = {Nucleic acids research}, volume = {43}, journal = {Nucleic acids research}, number = {6}, publisher = {Oxford Univ. Press}, address = {Oxford}, issn = {0305-1048}, doi = {10.1093/nar/gkv199}, pages = {3022 -- 3032}, year = {2015}, abstract = {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.}, language = {en} } @article{DelCampoBartholomaeusFedyuninetal.2015, author = {Del Campo, Cristian and Bartholom{\"a}us, Alexander and Fedyunin, Ivan and Ignatova, Zoya}, title = {Secondary Structure across the Bacterial Transcriptome Reveals Versatile Roles in mRNA Regulation and Function}, series = {PLoS Genetics : a peer-reviewed, open-access journal}, volume = {11}, journal = {PLoS Genetics : a peer-reviewed, open-access journal}, number = {10}, publisher = {PLoS}, address = {San Fransisco}, issn = {1553-7404}, doi = {10.1371/journal.pgen.1005613}, pages = {23}, year = {2015}, abstract = {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.}, language = {en} } @article{RoethleinMiettinenBorwankaretal.2014, author = {Roethlein, Christoph and Miettinen, Markus S. and Borwankar, Tejas and Buerger, Joerg and Mielke, Thorsten and Kumke, Michael Uwe and Ignatova, Zoya}, title = {Architecture of polyglutamine-containing fibrils from time-resolved fluorescence decay}, series = {The journal of biological chemistry}, volume = {289}, journal = {The journal of biological chemistry}, number = {39}, publisher = {American Society for Biochemistry and Molecular Biology}, address = {Bethesda}, issn = {0021-9258}, doi = {10.1074/jbc.M114.581991}, pages = {26817 -- 26828}, year = {2014}, abstract = {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.}, language = {en} } @article{WeissenbornIgnatovOcheletal.2014, author = {Weissenborn, Christine and Ignatov, Tanja and Ochel, Hans-Joachim and Costa, Serban Dan and Zenclussen, Ana Claudia and Ignatova, Zoya and Ignatov, Atanas}, title = {GPER functions as a tumor suppressor in triple-negative breast cancer cells}, series = {Journal of cancer research and clinical oncology : official organ of the Deutsche Krebsgesellschaft}, volume = {140}, journal = {Journal of cancer research and clinical oncology : official organ of the Deutsche Krebsgesellschaft}, number = {5}, publisher = {Springer}, address = {New York}, issn = {0171-5216}, doi = {10.1007/s00432-014-1620-8}, pages = {713 -- 723}, year = {2014}, abstract = {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.}, language = {en} } @article{MiettinenMonticelliNedumpullyGovindanetal.2014, author = {Miettinen, Markus S. and Monticelli, Luca and Nedumpully-Govindan, Praveen and Knecht, Volker and Ignatova, Zoya}, title = {Stable polyglutamine dimers can contain beta-hairpins with interdigitated side chains but not a-helices, alpha-nanotubes, beta-pseudohelices, or steric zippers}, series = {Biophysical journal}, volume = {106}, journal = {Biophysical journal}, number = {8}, publisher = {Cell Press}, address = {Cambridge}, issn = {0006-3495}, doi = {10.1016/j.bpj.2014.02.027}, pages = {1721 -- 1728}, year = {2014}, abstract = {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.}, language = {en} } @article{PuriWetzelSaffertetal.2014, author = {Puri, Pranav and Wetzel, Collin and Saffert, Paul and Gaston, Kirk W. and Russell, Susan P. and Varela, Juan A. Cordero and van der Vlies, Pieter and Zhang, Gong and Limbach, Patrick A. and Ignatova, Zoya and Poolman, Bert}, title = {Systematic identification of tRNAome and its dynamics in Lactococcus lactis}, series = {Molecular microbiology}, volume = {93}, journal = {Molecular microbiology}, number = {5}, publisher = {Wiley-Blackwell}, address = {Hoboken}, issn = {0950-382X}, doi = {10.1111/mmi.12710}, pages = {944 -- 956}, year = {2014}, abstract = {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.}, language = {en} } @article{GirstmairSaffertRodeetal.2013, author = {Girstmair, Hannah and Saffert, Paul and Rode, Sascha and Czech, Andreas and Holland, Gudrun and Bannert, Norbert and Ignatova, Zoya}, title = {Depletion of Cognate Charged Transfer RNA Causes Translational Frameshifting within the Expanded CAG Stretch in Huntingtin}, series = {Cell reports}, volume = {3}, journal = {Cell reports}, number = {1}, publisher = {Cell Press}, address = {Cambridge}, issn = {2211-1247}, doi = {10.1016/j.celrep.2012.12.019}, pages = {148 -- 159}, year = {2013}, abstract = {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.}, language = {en} } @article{BenteleSaffertRauscheretal.2013, author = {Bentele, Kajetan and Saffert, Paul and Rauscher, Robert and Ignatova, Zoya and Bluethgen, Nils}, title = {Efficient translation initiation dictates codon usage at gene start}, series = {Molecular systems biology}, volume = {9}, journal = {Molecular systems biology}, number = {6}, publisher = {Nature Publ. Group}, address = {New York}, issn = {1744-4292}, doi = {10.1038/msb.2013.32}, pages = {10}, year = {2013}, abstract = {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.}, language = {en} } @article{CzechWendeMoerletal.2013, author = {Czech, Andreas and Wende, Sandra and Moerl, Mario and Pan, Tao and Ignatova, Zoya}, title = {Reversible and rapid transfer-RNA deactivation as a mechanism of translational repression in stress}, series = {PLoS Genetics : a peer-reviewed, open-access journal}, volume = {9}, journal = {PLoS Genetics : a peer-reviewed, open-access journal}, number = {8}, publisher = {PLoS}, address = {San Fransisco}, issn = {1553-7404}, doi = {10.1371/journal.pgen.1003767}, pages = {9}, year = {2013}, abstract = {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.}, language = {en} } @article{LukoszekMuellerRoeberIgnatova2013, author = {Lukoszek, Radoslaw and M{\"u}ller-R{\"o}ber, Bernd and Ignatova, Zoya}, title = {Interplay between polymerase II- and polymerase III-assisted expression of overlapping genes}, series = {FEBS letters : the journal for rapid publication of short reports in molecular biosciences}, volume = {587}, journal = {FEBS letters : the journal for rapid publication of short reports in molecular biosciences}, number = {22}, publisher = {Elsevier}, address = {Amsterdam}, issn = {0014-5793}, doi = {10.1016/j.febslet.2013.09.033}, pages = {3692 -- 3695}, year = {2013}, abstract = {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.}, language = {en} } @article{VarshneyKumarIgnatovaetal.2012, author = {Varshney, Nishant Kumar and Kumar, R. Suresh and Ignatova, Zoya and Prabhune, Asmita and Pundle, Archana and Dodson, Eleanor and Suresh, C. G.}, title = {Crystallization and X-ray structure analysis of a thermostable penicillin G acylase from Alcaligenes faecalis}, series = {Acta crystallographica : Section F, Structural biology communications}, volume = {68}, journal = {Acta crystallographica : Section F, Structural biology communications}, number = {3}, publisher = {Wiley-Blackwell}, address = {Malden}, issn = {1744-3091}, doi = {10.1107/S1744309111053930}, pages = {273 -- 277}, year = {2012}, abstract = {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.}, language = {en} }