Electronic Thesis and Dissertation Repository

Thesis Format

Monograph

Degree

Master of Science

Program

Biochemistry

Supervisor

O'donoghue, Patrick

2nd Supervisor

Brandl, Christopher J

Co-Supervisor

Abstract

Mistranslation is an error in protein synthesis whereby the amino acids specified by the genetic code are misplaced by others in the growing polypeptide chain. The anticodon of tRNALeu can be altered allowing for the misincorporation of leucine at non-leucine codons. Observing the effect of tRNALeu variants on the viability of yeast and mammalian cells will provide information on their ability to cause mistranslation and potential relationship to genetic diseases. To explore this, a random pool of tRNALeu anticodonvariants was expressed in Saccharomyces cerevisiae. Furthermore, three mutant tRNALeu already sequenced from humans were expressed in murine Neuro 2A blastoma cells. There was slow growth in yeast when tRNALeu with proline (NGG) and arginine (GCG) anticodons were expressed. In N2a cells tRNALeu GAA lowered protein synthesis while tRNALeuCCA increased it. The effect of each tRNALeu anticodon variant varied depended on the anticodon with no obvious correlation to known biological factors.

Summary for Lay Audience

DNA is the genetic library of our cells. It stores all the essential data for creating proteins and other biological materials that allow for cells to function. To translate DNA into protein, it first must be transcribed into messenger RNA (mRNA). A protein called RNA polymerase II runs along DNA and adds nucleotides until an mRNA chain is created. This mRNA is delivered a macromolecular complex called the ribosome. A molecule known as Eukaryotic translation factor 1A (eEF1A) delivers tRNAs to the ribosome. tRNAs are another type of RNA which deliver amino acids, the building blocks of proteins, to the ribosome to allow protein synthesis.

This study endeavors to change a component of tRNAs called the anticodon to leucine carrying tRNAs. The anticodon is located at the bottom of the tRNA molecule and can base-pair with specific three nucleotide sequences in mRNA known as codons. Through changing the anticodon, the leucine carrying tRNA (tRNALeu) can bind to non-leucine codons and add leucine to a protein instead of another amino acid. This creates an abnormal protein which is likely harmful to cells but could provide benefits under certain circumstances. tRNALeu is special as they can change their anticodon and stilly carry its cognate (leucine) amino acid.

It is important to study the effects of the tRNALeu anticodon mutants as they have been found in some humans. Therefore, it is important to observe the effects of these tRNAs on yeast and mammalian cells to relate the data collected to human disease. Using these tRNAs to create abnormal proteins could help in engineering synthetic proteins with unique functions, or in making a codon ambiguously decode two amino acids. The results of this study show that certain tRNALeu variants with proline and arginine anticodons cause yeast cells to grow very slow. There are also human tRNALeu variants that mistranslate phenylalanine and methionine. These tRNAs either protein synthesis or increase toxicity when protein degradation is halted.

Download

Share

COinS