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CHARACTERIZING RNA TRANSCRIPTION AND DNA REPLICATION VIA RAMAN CRYSTALLOGRAPHY

Antonopoulos, Ioanna H
http://rave.ohiolink.edu/etdc/view?acc_num=case1428076280

Abstract Details

2015, Doctor of Philosophy, Case Western Reserve University, Biochemistry.
Nucleic acid polymerases are responsible for catalyzing DNA replication and transcription in all life domains. As the enzymatic activities of DNA and RNA polymerases govern the processes of gene duplication and gene expression, it is essential that we form a thorough understanding of these mechanisms. It is the goal of this thesis to address the structural dynamics and kinetics of nucleic acid polymerases, Thermus thermophilus RNA polymerase and RB69 DNA polymerases, by applying a Raman microscopic study to the nucleotidyl transfer reactions in single crystals. Here we provide Raman crystallographic evidence and kinetics for cognate and non-cognate ribonucleotide incorporation in single crystals of a bacterial RNA polymerase elongation complex from the thermophilic enzyme, Thermus thermophilus (Tth). Raman spectroscopic results show major changes associated with protein and DNA conformational changes, especially during non-cognate base misincorporation and are supported by polyacrylamide gel electrophoresis from a collaborating laboratory. Also, Raman data also suggest major conformational changes associated with nucleotide incorporation in single crystals of a RB69 DNA polymerase ternary complex, such as protein amide features, tyrosine ring modes, and nucleic acid base modes. Overall, these Raman observations report on the structural changes of enzyme-DNA interactions nucleic acid polymerase crystals and provide the opportunity to bridge X-ray crystallographic studies with solution studies. This is not only vital from a basic science perspective but also from a therapeutic standpoint as elucidating the structural basis for nucleic acid polymerase activity is key to designing therapeutics which can specifically target the RNA polymerase transcription or DNA polymerase replication machineries. As bacterial resistance continues to plague the world population, elucidating the effects of bactericidal antibiotics such as streptolydigin and tagetitoxin which uniquely target the nucleotide addition cycle of bacterial RNA polymerases, while leaving eukaryotic RNA polymerases essentially unaffected, is extremely important in treating bacterial infections. Similarly, understanding the replication mechanism and structure of viral DNA polymerases is of utmost importance in designing therapeutics against viruses such as the Herpes Simplex Virus, since the latter shows strikingly similar structural conservation to the RB69 DNA polymerase studied herein.
Paul Carey (Advisor)
Vivien Yee (Committee Chair)
Pieter deHaseth (Committee Member)
Michael Harris (Committee Member)
Derek Taylor (Committee Member)
163 p.
Biochemistry
Raman spectroscopy; transcription elongation; Thermus thermophilus RNA polymerase

Recommended Citations

Citations

  • Antonopoulos, I. H. (2015). CHARACTERIZING RNA TRANSCRIPTION AND DNA REPLICATION VIA RAMAN CRYSTALLOGRAPHY [Doctoral dissertation, Case Western Reserve University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=case1428076280

    APA Style (7th edition)

  • Antonopoulos, Ioanna. CHARACTERIZING RNA TRANSCRIPTION AND DNA REPLICATION VIA RAMAN CRYSTALLOGRAPHY. 2015. Case Western Reserve University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=case1428076280.

    MLA Style (8th edition)

  • Antonopoulos, Ioanna. "CHARACTERIZING RNA TRANSCRIPTION AND DNA REPLICATION VIA RAMAN CRYSTALLOGRAPHY." Doctoral dissertation, Case Western Reserve University, 2015. http://rave.ohiolink.edu/etdc/view?acc_num=case1428076280

    Chicago Manual of Style (17th edition)

case1428076280
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© 2015, all rights reserved.
This open access ETD is published by Case Western Reserve University School of Graduate Studies and OhioLINK.