This dissertation covers two projects linked by their involvement in the modification of tRNA bases.
The first project focused on an investigation of a role for the modified base Archaeosine, the ubiquitous modification in tRNA in the archaeal domain. Initial work was performed on a set of in vitro prepared tRNA modified to feature either the canonical guanine base at position 15, preQ0 (TGT product) or Archaeosine (ArcS product). There was very little difference in the thermal stability of tRNAs containing these modifications in the halophilic H. volcanii tRNASer or E. coli tRNAGln. In tRNAGln taken from M. thermautotrophicum however, there was a 2°C increase in melting point in 50 mM MgCl2 upon modification to archaeosoine.
Benefitting from the development of genetic tools for the generation of specific deletion mutants of the thermophile Thermococcus kodakarensis, it was possible to start investigation of tRNAs that have been hypomodified in vivo due to the lack of arcTGT (TK0760) and ArcS (TK2156). In vitro modified equivalents of the GlnCUG isoacceptor were also prepared. Thermal stability of these tRNAs show virtually identical melting transitions, with a biphasic denaturation occurring at all magnesium concentrations tested. Isolation of the CUG isoacceptor from the in vivo maturated total tRNA pool allowed melts of specifically hypomodified tRNAs. Those containg Archaeosine (WT) and genetically encoded guanine (∆tgt) showed identical melting profiles with Tm beyond the 98°C limit of the experiment. In the preQ0 containing in vivo RNA the shows a lag in its magnesium response, and a more persistent biphasic melting profile. At 10mM Mg2+ concentration the preQ0 containing tRNA is approaching a Tm of 98°C though the turn over point in the melt is not well defined.
The second project was to investigate the product of base treatment of the oxidized cofactor NAD(P)+. This cofactor is involved in the biosynthesis of preQ1 from preQ0 in bacterial systems and at low concentrations it can be difficult to quantify enzyme activity based on direct quantitation. Under these conditions a fluorescence based method where by the production of NAD(P)+ is measured rather than the consumption of NAD(P)H.
Base treatment of the oxidized cofactor generates a fluorescent species with an efficiency of 95%. The assay has been used extensively by our group to track activity of various enzymes including QueF, however the identity of the fluorophore had not been established. Purification of the fluorescent product was achieved by isocratic HPLC in water using a reverse phase column. It was found that the assay conditions previously used (7.5M NaOH for 2 hours) were actually counterproductive for maximizing fluorescence yield. Incubation at 2M NaOH gave a 35% increase in product yield. The isolated product was determined to have molecular weight of 123.0318 (3.6 ppm by accurate mass ESI MS). 1H and 13C NMR were used to confirm the structure to be that of 2-hydroxynicotinaldeyde. It was also possible to determine the quantum yield for the molecule to 0.11. Work carried out previously on pyridinium based NADP analogs is consistent with the identity of the fluorophore presented here.
| Identifer | oai:union.ndltd.org:pdx.edu/oai:pdxscholar.library.pdx.edu:open_access_etds-4655 |
| Date | 08 June 2017 |
| Creators | Turner, Ben |
| Publisher | PDXScholar |
| Source Sets | Portland State University |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Dissertations and Theses |
Page generated in 0.0023 seconds