Insights into the establishment and continuing evolution of the genetic code

The precise mechanisms underlying the evolution of the genetic code have eluded researchers for decades. The structure and order of the code suggests a biological, chemical, or physical impetus directing the codon assignments, however these forces remain mysterious. In addition, other forces continue to shape the code, generating natural and unnatural variants of the canonical genetic code. My work is split between these two distinct avenues or research : 1) Statistical evidence from ribosome structures suggests that a certain subset of amino acids were assigned to their codons via a stereochemical interaction with their respective anticodons. In addition, a novel in vivo system relying on the ambiguous decoding of isoleucine in E. coli suggests that mRNA sequence context outside of the A-site can influence tRNA selection in the ribosome. The underlying mechanism for this influence may be rooted in stereochemical interactions, as anticodons dominate these contextual moieties. 2) Variant codes can give valuable and rare insights into the continued evolution of the genetic code. Mimicking these types of variation in the lab will grant a valuable tool for this field, however full reassignment has yet to be achieved. Two E. coli strains were generated (JX1.0 and JX3.0) that harbor no genomic copy of release factor one. The UAG codon is now nonsense in these strains, and no longer encodes for a stop signal. In addition, the expression of an unnatural suppression system leads to efficient suppression of the UAG with an unnatural amino acid, and represents the first engineered organism harboring such a reassignment. Suppression of multiple UAG codons in a single protein is efficient and overcomes a major technological hurdle in the field. These advances will leave a lasting imprint on these fields, and will help to further understand the evolution of the genetic code. In addition, the novel techniques, strains, and findings described herein may help to answer many remaining questions regarding both the early and late events in the evolution of the genetic cod