Aaron Sykes is an undergraduate researcher in Dr. Yale Goldman’s lab. He attends Villanova University.
In vitro Investigation of Eukaryotic Translation using Single-Molecule FRET
Nonsense mutations lead to approximately 7000 genetically transmitted disorders including Cystic Fibrosis and Duchenne Muscular Dystrophy. Nonsense mutations give rise to premature termination codons (PTC), which are replacements of an amino acid codon in mRNA by one of the three stop codons, and lead to inactive truncated protein products. Sometimes, translational readthrough occurs where selected near cognate tRNAs at the PTC position insert the corresponding amino acids into the new polypeptide, restoring the production of full length functional proteins. However, the specific molecular mechanisms by which this process occurs are still not well understood. Studies of readthrough using animals, intact cells, or cell extracts show a variety of mechanisms of readthrough, and so attempts to determine the precise mechanisms of action are complicated. To try to investigate the details of the mechanisms of readthrough that directly affect the ribosome pathway, single molecule ﬂuorescence resonance energy transfer (smFRET) on a highly puriﬁed, eukaryotic cell-free protein synthesis system is being developed. A critical component of this assay are the Glutamine and Tryptophan tRNAs labeled with fluorescent cyanine dyes, Cy3 and Cy5, that are used for the smFRET. To obtain large enough quantities for the assay, the optimization of the charging of these tRNAs is necessary. Different charging conditions were tested to optimize the charging conditions for Cy5-labeled Tryptophan tRNA including the incubation time of the charging reaction, concentration of ATP, pH of the Tris-HCl buffer solution, and concentration of tryptophanyl-tRNA synthetase. The amount of charging is then quantified by measuring the amount of ionizing radiation from the incorporation of a mixture of amino acids that are radioactive and non-radioactive. Preliminary results show that an incubation time of 10 minutes, buffer pH of 7.8, 10 mM of ATP, and equal volume of synthetase to tRNA yield the most amount of charged tRNA. Further work will be done to optimize these conditions and test their activity with an octapeptide synthesis assay and with the smFRET assay. Optimization of the preparation of the tRNA and the other components of this assay will allow the direct investigation of translation and translational readthrough on the single molecule level and lead to the development of new therapeutic agents to treat nonsense mutation related disorders.