Mechanism and Evolution of tRNA CCA-adding Enzymes Restricted; Files Only

Abstract

The nucleotidyltransferase superfamily (NTSF) contains enzymes characterized by their ability to add nucleotides onto a specific target. tRNA nucleotidyltransferases (TNTs) are a subfamily within the NTSF that have the ability to add nucleotides onto a transfer RNA (tRNA) in a template-independent manner. Specific TNTs, called CCA-adding enzymes, act on the 3’ end of tRNA to catalyze the addition of the CCA end and are present within all domains of life. The 3’ CCA end is necessary for the tRNA molecule to be aminoacylated, after which the tRNA can deliver an amino acid to the ribosome during the elongation phase of translation. However, the CCA end is often removed during tRNA maturation or can become damaged by multiple cellular enzymes, so CCA-adding enzymes are essential for the cell. There are two main mechanisms by which the CCA ends are added to the 3’ of tRNAs. In the first mechanism, a single enzyme adds two cytosines (C) and a single adenosine (A). In the second mechanism, two enzymes are required, one to add the two C nucleotides and the other to add the terminal A nucleotide. It is unknown which mechanism evolved first. To address this issue, a most recent common ancestor (FPMRCA) sequence was predicted using the program FireProt^ASR. In this thesis, I analyzed the AlphaFold-predicted model of FPMRCA and determined that the structure is likely similar to other CCA-adding enzymes. To test their function, I began studies to optimize overexpression and purification of recombinant FPMRCA. The goal is to ultimately perform a CCA-adding enzymatic assay to compare its function to canonical CCA-adding enzymes that will shed light onto the evolutionary pathway by which CCA-, CC- and A- adding enzymes evolved.

Table of Contents

1. Introduction ............................................................................................................................................... 1

        1.1 The Central Dogma of Life and Translation ........................................................................................... 1

        1.2 The importance of the CCA end of tRNA molecules ............................................................................... 2

        1.3 The divergent evolution of CCA-adding enzymes .................................................................................. 4

2. Materials and Methods ................................................................................................................................ 9

        2.1 Strains and Plasmids ........................................................................................................................... 9

        2.2 Optimization of Induction and Expression .......................................................................................... 10

        2.3 Protein Purification ........................................................................................................................... 11

        2.4 Molecular Cloning ............................................................................................................................. 13

                  2.4.1 PCR Amplification .................................................................................................................. 13

                  2.4.2 Restriction Enzyme Digestions ................................................................................................ 16

                  2.4.3 Ligation Reactions and Transformations .................................................................................. 16

                  2.4.4 Transformations and Colony PCR ............................................................................................ 17

                  2.4.5 pCOLD Induction Trial ............................................................................................................ 17

        2.5 AlphaFold Structure Prediction .......................................................................................................... 18

3. Results and Discussion .............................................................................................................................. 18

        3.1 Structural Analysis ............................................................................................................................ 18

        3.2 Protein Induction, Expression and Purification .................................................................................... 20

        3.3 Molecular Cloning ............................................................................................................................. 24

4. Conclusion and Future Directions ............................................................................................................... 24

5. References ................................................................................................................................................ 27

About this Honors Thesis

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School	Emory College
Department	Chemistry
Degree	B.S.
Submission	Honors Thesis
Language	English
Research Field	Chemistry, Biochemistry Biology, Evolution and Development
Mot-clé	tRNA CCA-adding Enzymes Protein Structure and Function tRNA Nucleotidyltransferases
Committee Chair / Thesis Advisor	Christine M. Dunham, Ph.D., Emory University
Committee Members	Vincent Conticello, Ph.D., Emory University Katherine M. Davis, Ph.D., Emory University

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