Date of Award

Spring 2020

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

School

Mathematics and Natural Sciences

Committee Chair

Dr. Faqing Huang

Committee Chair School

Mathematics and Natural Sciences

Committee Member 2

Dr. Vijay Rangachari

Committee Member 2 School

Mathematics and Natural Sciences

Committee Member 3

Dr. Yanlin Guo

Committee Member 3 School

Biological, Environmental, and Earth Sciences

Committee Member 4

Dr. Douglas Masterson

Committee Member 4 School

Mathematics and Natural Sciences

Committee Member 5

Dr. Jacques Kessl

Committee Member 5 School

Mathematics and Natural Sciences

Abstract

The ability of RNA to store genetic information and to catalyze biochemical transformations led to the speculation of the existence of RNA world before the evolution of contemporary ribonucleoprotein (RNP) world. Recent discovery of RNA molecules containing metabolic cofactors including coenzyme A and its various thioesters at their 5’ end further supported the RNA world hypothesis as these CoA-linked RNA molecules could be the molecular fossils with very ancient origin. As both RNA and Coenzyme A are believed to have co-existed since last universal common ancestor (LUCA) or even before, the CoA-RNA conjugates in current biology may reveal fundamental molecular secrets involved in evolution. Furthermore, these CoA-RNA conjugates may not be just remnants of evolution, rather may have an important functional significance in contemporary metabolism and gene regulation. The successful characterization of these conjugates will expand our knowledge in RNA function and CoA function. However, the sequence, metabolism and biological role of these RNA species are still unknown. The aim of this study is to capture CoA-RNA sequences form E. coli total RNA to uncover their sequences and to investigate their biogenesis.

The successful characterization of CoA-RNA requires a specific protocol to capture them. The development of such protocol requires an easy access to synthetic CoA-RNA. While our lab previously developed a method to incorporate CoA into RNA co-transcriptionally by using dephospho CoA as a transcription initiator, the limited availability of dephospoCoA restricts an easy access to synthetic CoA-RNA. In the first section, a simple and easy method for the synthesis of dephospho CoA and its oxidized dimer was developed. Two enzymes of CoA biosynthetic pathway, PanK and PPAT were cloned in a single plasmid, and purified in a single enzyme preparation. The synthesis of dephsphoCoA was achieved by the enzyme cocktail and pure product was obtained by a simple reverse phase column chromatography. The method was extended further to synthesize various dephosphoCoA analogs including amino dephosphoCoA and biotin dephosphoCoA.

In the second section, two strategies were investigated for their application in CoA-RNA capture. In first strategy, various acyl-CoA ligases including acetyl CoA synthetase (ACS), Malonyl CoA synthetase (MatB), Succinyl CoA synthetase (SucCD), medium chain fatty acyl CoA synthetase (FadK) and Long chain fatty acyl CoA synthetase were cloned, expressed and tested their ability to accept biotinylated fatty acid and CoA-RNA as substrates. In the second strategy, various pantetheine and phosphopantetheine analogs with biotin as a purification handle, [14-C] acetate as a reporter tag, and variable number of positive charges to mediate cellular uptake were synthesized. During the synthesis of such analogs, a novel method for the selective protection of secondary alcohols in presence of their primary counterparts was developed.

In the third section of this work, a mechanism of CoA-RNA biogenesis was explored. Synthesized phosphopantetheine analog was used to investigate whether CoA-RNA can be generated post-transcriptionally. We found that a CoA biosynthetic enzyme, PPAT (coaD), can accept ATP-RNA as its substrate and yields CoA-RNA. our results showed that the suitability of ATP initiated RNA to serve as a PPAT substrate is determined by its 5’ structure. An RNA having at least four unstructured nucleotides at the 5’ end can participate in PPAT catalyzed phosphopantetheine transfer reaction. Furthermore, the rate of the reaction was independent to the number of 5’ unstructured nucleotides at least for the range of 4-10. These findings established the post-transcriptional transfer of 4’-phosphopantetheine to ATP-RNA as another mode of CoA-RNA biogenesis besides previously characterized RNAP mediated co-transcriptional incorporation.

Collectively, we have developed an easy and simple method to prepare dephospho CoA and its analogs, investigated chemo-enzymatic strategies for CoA-RNA capture and, discovered a post-transcriptional mechanism of CoA-RNA biogenesis. Our results suggest that the existence of CoA-RNA in extant biology may be evolutionary. Future studies may lead to development of specific CoA-RNA capture and will expand our understandings on how ribozyme catalysis of the RNA world was transformed into protein enzyme-based catalysis of contemporary world.

ORCID ID

https://orcid.org/0000-0002-3985-0980

Available for download on Friday, May 14, 2021

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