Orthogonal Protein Synthesis by Ribosomes with an Integrated mRNA

Ribosomes drive protein synthesis by translating the mRNA into the amino acid sequence of proteins. Given the importance of proteins and peptides, many efforts have focused on engineering the ribosome to expand its functionality beyond its repertoire of only 20 naturally occurring proteinogenic amino acids. However, developing such systems faces the challenge of overcoming the detrimental effects that most ribosomal modifications impose upon the ability of the engineered ribosomes to synthesize cellular proteins. One way to circumvent this limitation is to functionally isolate the population of engineered ribosomes from the rest, thus generating two pools of ribosomes in the same cell: (I) wildtype ribosomes translating endogenous mRNAs and (II) specialized ribosomes translating their designated mRNAs. To develop a translation system with functional separation with respect to wildtype ribosomes, we have designed a new ribosome in which the mRNA is physically tethered to the rRNA of the small ribosomal subunit. We provide evidence that these engineered ribosomes are highly specialized, indicating that their attached mRNA is translated predominantly in cis. Furthermore, we successfully integrated this design into ribosomes with tethered subunits to expand functional isolation to the large ribosomal subunit. This ribosome-mRNA design demonstrates the viability of a fully integrated translation system whose specialization can theoretically allow the assignment of individual ribosomes to the synthesis of specific polypeptides, including those containing non-natural or difficult-to-translate amino acid sequences. The results of our work open new opportunities for synthetic biology.

Author dc:creator

• Kasra Alizadeh (23292040)

• Biology, Molecular
• Biology, Microbiology

Statement dc:rights

• In Copyright
• Open Access after 2028-01-01

DOI dc:identifier

https://doi.org/10.25417/uic.31451716.v1

OAI identifier oai:identifier

oai:figshare.com:article/31451716