Misreading errors are one type of missense error that can occur during translation that result in one amino acid substituting for another. They are the result of a mismatch between a codon and the anticodon of a near-cognate tRNA in the aminoacyl (A) site of the ribosome during the decoding of a mRNA and the subsequent misincorporation of the incorrect amino acid into the peptide.

I am studying misreading errors in vivo during the translation of the firefly luciferase reporter gene. There is a lysine residue at position 529 of firefly luciferase that is conserved and is essential for luciferase activity. I use site-directed mutagenesis to mutate this AAA lysine codon to all possible single base mutations in each of the codon positions, which represent single nucleotide mismatches with the UUU anticodon of the isoaccepting lysine-tRNA(UUU). I express the mutant luciferase enzymes in E. coli and assay for luciferase activity, which indicates some degree of misreading of the mutated codons by lys-tRNA(UUU) and incorporation of lysine into the protein. In addition, I will overexpress the lys-tRNA(UUU) in the cells, and I expect to see increases in luciferase activity, which would support the idea that activity is due to misreading. Finally, I will attempt to correlate the misreading error frequencies at the three codon positions with the structure of the standard genetic code. This research could provide experimental support for the adaptive theory of genetic code evolution, which suggests that the code evolved to minimize the effects of mutational and translational errors.