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SPECIFICITY LANDSCAPE OF RIBONUCLEASE P PROCESSING OF PRE-TRNA SUBSTRATES BY HIGH-THROUGHPUT ENZYMOLOGY

Niland, Coutrney Nicole
http://rave.ohiolink.edu/etdc/view?acc_num=case1479394526204776

Abstract Details

2017, Doctor of Philosophy, Case Western Reserve University, Biochemistry.
To fully understand the roles of RNA processing enzymes in cellular processes and human health, it is essential to dissect their substrate specificity. Using Ribonuclease P (RNase P) as a model system, this body of work seeks to better understand multiple substrate recognition by RNA processing enzymes. The ubiquitous endonuclease RNase P removes the 5' leader from all pre-tRNAs in the cell. To understand how variation in the 5' leader is accommodated by the active site of RNase P, we comprehensively determined the processing rates of pre-tRNA substrates with all possible sequence combinations in the 5' leader. This quantification involved Illumina(R) sequencing of the residual substrate population at different reaction times to monitor substrate depletion and calculate relative rate constants, a technique termed HTS-Kin. Additionally, the 5' leader of pre-tRNA is recognized by both the catalytic RNA subunit (P RNA) and smaller protein subunit of RNase P, C5. We therefore hypothesized that variation in substrate sequence contacting one enzyme subunit may alter the recognition or energetic contribution of contacts to the other enzyme subunit. Upon comprehensive determination of RNase P specificity for 5' leader sequences, we have determined that this enzyme is tuned for specificity at association as the catalytic rate constant is unaffected by substrate variation. We have also performed mechanistic analysis to identify the key sources of error in the HTS-Kin technique: experimental error and Illumina sequencing error. Finally, we ascertained that the sequence identity of 5' leader nucleotides contacting P RNA does not alter C5 protein specificity but rather modulates its energetic contribution to the processing rate.
Michael Harris, Ph.D. (Advisor)
Hung-Ying Kao, Ph.D. (Committee Chair)
Eckhard Jankowsky, Ph.D. (Committee Member)
Blanton Tolbert, Ph.D. (Committee Member)
Michael Weiss, M.D., Ph.D. (Committee Member)
163 p.
Biochemistry
RNase P, High-Throughput Sequencing, Enzymology, Enzyme Specificity, Molecular Recognition

Recommended Citations

Citations

  • Niland, C. N. (2017). SPECIFICITY LANDSCAPE OF RIBONUCLEASE P PROCESSING OF PRE-TRNA SUBSTRATES BY HIGH-THROUGHPUT ENZYMOLOGY [Doctoral dissertation, Case Western Reserve University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=case1479394526204776

    APA Style (7th edition)

  • Niland, Coutrney. SPECIFICITY LANDSCAPE OF RIBONUCLEASE P PROCESSING OF PRE-TRNA SUBSTRATES BY HIGH-THROUGHPUT ENZYMOLOGY. 2017. Case Western Reserve University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=case1479394526204776.

    MLA Style (8th edition)

  • Niland, Coutrney. "SPECIFICITY LANDSCAPE OF RIBONUCLEASE P PROCESSING OF PRE-TRNA SUBSTRATES BY HIGH-THROUGHPUT ENZYMOLOGY." Doctoral dissertation, Case Western Reserve University, 2017. http://rave.ohiolink.edu/etdc/view?acc_num=case1479394526204776

    Chicago Manual of Style (17th edition)

case1479394526204776
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This open access ETD is published by Case Western Reserve University School of Graduate Studies and OhioLINK.