Code Within Codes: Codon Usage Regulate Protein Expression, Structure, and Function

Most amino acids are encoded by two to six synonymous genetic codons. Synonymous codons are not used with the same frequency in all organisms, and every organism has its own preferred codon usage bias. Codon usage bias has been shown to positively correlate with tRNA abundance, thus optimal codons are thought to be translated more efficiently and accurately. Consistent with this, strong codon usage biases have been shown to be important for the expression of highly expressed genes in different organisms, and codon optimization has been widely used to enhance heterologous protein expression. Therefore, codon usage can be an important determinant in gene expression. In addition, codon usage has been shown to influence translation elongation rate and protein structure by affecting the co-translational folding process in E. coli, fungi, and insects. In addition to its role in regulating protein translation, codon usage also has a major role in determining the level of gene expression through transcriptional and post-transcriptional processes. As such, gene codon usage has been proposed to be a code within the genetic code that can determine both gene expression levels and protein structures and therefore activity. However, the effects of codon usage in multi-tissue organisms, for example, animals and humans, are not clear. In the first part of the thesis, by codon-optimizing open reading frame of Drosophila period gene, I showed that dper codon usage is critical for its circadian clock function. Optimization of dper codon usage resulted in conformational changes of dPER protein, altered dPER phosphorylation profile and stability, and impaired dPER repressor function in the circadian negative feedback loop. In the second part of the thesis, I reported that changing rare codons to common in KRAS increased translation and mRNA levels. Regulation of mRNA levels is a major mechanism affecting KRAS levels, but the effect was not a product of mRNA stability, but instead transcriptional. Moreover, codon usage also had an impact on the structure of KRAS. Thus, the rare codon bias of KRAS effects more aspects of protein production and function than previously appreciated, which has important implications for other rare codon enriched mammalian genes.