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Quantitative Modeling of Protein - Nucleic Acid Interactions

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2018, Doctor of Philosophy, Ohio State University, Biophysics.
We develop quantitative models to study the mechanical properties of nucleic acid-protein complexes, including the nucleosome and the ribosome, and their interactions with the surrounding environment. Such interactions are known to be involved in many different cellular processes, such as gene transcription and translation. Detailed biophysical models of these functional complexes can thus provide mechanical insights and quantitative measurements for such cellular processes, which may not be easily learned from wet lab experiments alone. We thus believe that our contribution to the field can greatly facilitate future research on nucleic acid-protein complexes interactions. First, we develop quantitative models of the ribosome and apply this model, in combination with high-throughput ribosome profiling data, to study the interaction between ribosome and nuclease. Ribosome profiling has been widely used to study translation in a genome-wide fashion. It requires deep sequencing of ribosome protected mRNA fragments followed by mapping of the mRNA fragments to the reference genome. For applications such as identification of ribosome pausing sites, it is not enough to map an mRNA fragment to a given gene, but the exact position of the ribosome represented by the fragment must be identified for each mRNA fragment. The assignment of the correct ribosome position is complicated by the broad length distribution of the ribosome protected fragments caused by the known strong sequence biases of micrococcal nuclease (MNase), the most widely used nuclease for digesting mRNAs in bacteria, such as \emph{E. coli}. Available mapping algorithms, such as the fixed offset method, the length dependent offset method and the center weighted method suffer from either MNase bias or low accuracy in characterizing the ribosome pausing kinetics. In this thesis, we introduce a new computational method for mapping the ribosome protected fragments to ribosome locations. We first develop a mathematical model of the interplay between MNase digestion and ribosome protection of the mRNAs. We then use the model to reconstruct the ribosome occupancy profile on a per gene level. We demonstrate that our method has the capability of mitigating the sequence bias introduced by MNase and accurately locating ribosome pausing sites at codon resolution. We believe that our method can be broadly applied to ribosome profiling studies on prokaryotes where codon resolution is necessary. Second, we develop quantitative models of the nucleosome and study its asymmetric wrapping behavior. In eukaryotic cells, DNA is packaged into chromatin where nucleosomes are the basic packaging unit. Important cellular processes including gene expression, DNA replication, and DNA repair require nucleosomal DNA to be unwrapped so that functional proteins can access their target sites, which otherwise are sterically occluded. A key question in this process is what are the unwrapped conformations individual nucleosomes adopt within the context of chromatin. Here, we develop a concomitant nucleosome unwrapping model to address this question. We hypothesize that for a given end-to-end distance of the nucleosomal DNA, the nucleosomal DNA stochastically unwraps from the histone core from both ends independently and that this combination of unwrapping from both sides results in a significant increase in the distance between the DNA extending from both sides of the nucleosomes. We test our model on recently published experiments using a DNA origami nanocaliper that quantifies nucleosome unwrapping (Le \emph{et al.}, 2016) and achieve good agreement between experiment and model prediction. We then use our model to investigate the DNA origami caliper distribution when attached to a hexasome (a nucleosome lacking a H2A/H2B dimer). We predict a significant shift in the caliper angle distribution because of the asymmetric structural features of the hexasome, which we then verify by comparison to experiments. Our model, supported by current experiments, appears to be broadly applicable to studies of nucleosome dynamics, chromatin dynamics, and regulatory processes involving nucleosome unwrapping, as well as to optimization of future studies of the mechanical properties of other biomolecules using DNA origami structures. Third, we develop quantitative models for an extended type of DNA origami caliper used in the previous nucleosome-nanocaliper construct. The new DNA origami caliper has multiple complementary single stranded DNAs extending out of the main body. The hybridization of the single stranded DNAs can provide a way to control the actuation of the caliper. By calibrating the model to the experimental system, we aim to guide the design and implementation of these DNA origami nano devices and save the efforts on trial and error by providing predictions of outcomes of experiments.
Ralf Bundschuh (Advisor)
Michael Poirier (Committee Member)
Carlos Castro (Committee Member)
William Ray (Committee Member)
113 p.

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Citations

  • Zhao, D. (2018). Quantitative Modeling of Protein - Nucleic Acid Interactions [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1534755895588153

    APA Style (7th edition)

  • Zhao, Dengke. Quantitative Modeling of Protein - Nucleic Acid Interactions. 2018. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1534755895588153.

    MLA Style (8th edition)

  • Zhao, Dengke. "Quantitative Modeling of Protein - Nucleic Acid Interactions." Doctoral dissertation, Ohio State University, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=osu1534755895588153

    Chicago Manual of Style (17th edition)