Cys-tRNAj as a Second Translation Initiator for Priming Proteins with Cysteine in Bacteria

Area of Science:

• Synthetic Biology
• Molecular Biology
• Genetic Engineering

Background:

• Genetic translation relies on specific initiator tRNAs for protein synthesis.
• Controlling protein expression is crucial for various biological applications.

Purpose of the Study:

• To develop an alternative protein priming system in Escherichia coli.
• To enable N-terminal cysteine incorporation at non-canonical codons.

Main Methods:

• Designed a chimeric initiator tRNA (Cys-tRNAj) combining features of elongator tRNA^Cys and initiator tRNAfMet.
• Utilized a selection strategy based on N-terminal cysteine requirement for glucosamine-6-phosphate synthase activity.
• Validated the system in vitro using luciferase assays and in vivo using thymidylate synthase expression.

Main Results:

• Successfully engineered a Cys-tRNAj capable of initiating translation with N-terminal cysteine.
• Demonstrated biochemical evidence of cysteine coding at the protein priming stage.
• Showed initiation of translation at non-UGC codons using the engineered tRNAj.

Conclusions:

• The developed system allows recoding of protein priming in E. coli with controlled expression.
• This advancement opens possibilities for evolving xenonucleotides and tXNAj in vivo.