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Fluor-labeling of RNA and Fluorescence-based Studies of Precursor-tRNA Cleavage by Escherichia coli Ribonuclease P

Wallace, Andrew J
http://rave.ohiolink.edu/etdc/view?acc_num=osu1374489993

Abstract Details

2013, Master of Science, Ohio State University, Biochemistry.
RNase P participates in tRNA biogenesis by removing the 5'-leader of precursor-tRNAs (pre-tRNAs). A possible relic of the RNA world, the ribonucleoprotein form of RNase P performs pre-tRNA processing using an RNA catalyst, associated with a different number of essential protein cofactors in the three domains of life. In addition to being a paradigm for studies of protein-aided RNA catalysis, there is some interest in exploiting structural differences between the bacterial and eukaryotic variants to develop new antibacterial drugs. Facile high-throughput assays would aid in the identification of such agents. Since its discovery four decades ago, RNase P activity has typically been assessed by monitoring the cleavage of internally or terminally 32P-labeled pre-tRNAs. Following denaturing polyacrylamide gel electrophoresis to resolve the substrate and products, the extent of cleavage is quantitated using a phosphorimager. Fluor-labeled pre-tRNAs provide an attractive non-radioisotopic alternative while retaining high sensitivity and offering the potential for high-throughput assays. With some modifications to the copper(I)-mediated alkyne-azide cycloaddition, we have generated at low cost 5'-fluor-labeled pre-tRNAs. An in vitro transcription primed with 5'-azido-5'-deoxyguanosine (AzG) yielded a 5'-azide-bearing pre-tRNA, which was then coupled to either a homemade alkyne-bearing carboxyfluorescein derivative or commercially available alkyne derivatives of Cy3 (Cyanine 3, where 3 refers to the number of carbon atoms in the polyethylene region) and Alexa Fluor® 647. I have assessed the extent of AzG incorporation at the 5'-terminus and found it to be 87 ± 2% in a model RNA tetramer, with the remainder accounted for by GTP, and I have found the extent of fluor labeling to be 65% in a pre-tRNA substrate. My experiments revealed that several such economically prepared fluor-labeled pre-tRNAs are cleaved by in vitro reconstituted Escherichia coli RNase under multiple-turnover conditions accurately and with efficiency similar to that of radio-labeled pre-tRNAs. Moreover, the fluor-labeled pre-tRNA and products could also be separated and quantitated by laser-induced fluorescence capillary electrophoresis (LIF-CE) in an automated DNA sequencer (ADS). LIF-CE with an ADS¬¬ not only allows the assay to be high-throughput, but also enables facile detection of small amounts of mis-cleavage products when non-consensus pre-tRNAs are processed by RNase P. An advantage with fluor-labeled pre-tRNAs, not afforded by radiolabeled counterparts, is the ability to assay in the same reaction two or more substrates of the same length but different composition, an important consideration for substrate-recognition studies. This new approach will provide an experimental handle to better understand the rates at which RNase P processes various pre-tRNAs in vivo, an aspect that remains to be fully investigated.
Venkat Gopalan, PhD (Advisor)
Edward J. Behrman, PhD (Committee Member)
115 p.
Biochemistry
RNase P; Ribonuclease P; Click Chemistry; high-throughput screening

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Citations

  • Wallace, A. J. (2013). Fluor-labeling of RNA and Fluorescence-based Studies of Precursor-tRNA Cleavage by Escherichia coli Ribonuclease P [Master's thesis, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1374489993

    APA Style (7th edition)

  • Wallace, Andrew. Fluor-labeling of RNA and Fluorescence-based Studies of Precursor-tRNA Cleavage by Escherichia coli Ribonuclease P . 2013. Ohio State University, Master's thesis. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1374489993.

    MLA Style (8th edition)

  • Wallace, Andrew. "Fluor-labeling of RNA and Fluorescence-based Studies of Precursor-tRNA Cleavage by Escherichia coli Ribonuclease P ." Master's thesis, Ohio State University, 2013. http://rave.ohiolink.edu/etdc/view?acc_num=osu1374489993

    Chicago Manual of Style (17th edition)

osu1374489993
597
© 2013, some rights reserved.Creative Commons License Fluor-labeling of RNA and Fluorescence-based Studies of Precursor-tRNA Cleavage by Escherichia coli Ribonuclease P by Andrew J Wallace is licensed under a Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported License. Based on a work at etd.ohiolink.edu.
This open access ETD is published by The Ohio State University and OhioLINK.