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 - 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 - 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 - 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 -