Measuring and modifying translation in Escherichia coli through start codon and orthogonal tRNA engineering

Vincent RM1, Wright BM1, Hecht A2,3,4, Glasgow J2,3,4, Bawazer L2,3,4, Munson MS2,3,4, Cochran JR2,4, Endy D2,4, Salit M2,3,4 and Jaschke PR1

  1. Department of Molecular Sciences, Macquarie University, Sydney, NSW.
  2. Joint Initiative for Metrology in Biology, Stanford, USA.
  3. Genome-scale Measurements Group, National Institute of Standards and Technology, Stanford, USA.
  4. Department of Bioengineering, Stanford, USA.

Efficient translation initiation in bacteria requires the specific interaction between the start codon on the mRNA and the anticodon on the initiator tRNA. Recently, we measured the interaction strength of E. coli initiator tRNA with all 64 possible start codons in vivo. We found a surprising number of non-canonical start codons leading to translation initiation. Based on these findings, we have been working towards creating an orthogonal system that specifically initiates translation at UAG codons. An interesting feature of the standard genetic code is the dual function of AUG as both the dominant initiation codon as well as a methionine codon internal to genes. The AUG codon duality can create issues in gene prediction and genetic design. For example, in recombinant protein design, in-frame AUG codons can behave as start codons if upstream sequence resembles a Shine-Dalgarno site. Therefore, an orthogonal translation initiation system using a codon uniquely reserved for translation initiation could overcome this problem as well as provide other engineering benefits. In the genetic code, UAG normally signals "stop translation". This UAG can be freed up in engineered strains where all native UAG codons are recoded to the UAA stop codon. Here, we will describe deployment of an orthogonal initiator tRNA within a UAG-less E. coli strain called C321 and effects on fitness and proteome.