Expanding the Scope of Protein Synthesis Using Modified Ribosomes

Abstract

The ribosome produces all of the proteins and many of the peptides present in cells. As a macromolecular complex comprised of both RNAs and proteins, it employs a constituent RNA to catalyze the formation of peptide bonds rapidly and with high fidelity. Thus the ribosome can be argued to represent the key link between the RNA World, in which RNAs were the primary catalysts, and present biological systems in which protein catalysts predominate. In spite of the well known phylogenetic conservation of ribosomal RNAs through evolutionary history, ribosomal RNAs can be altered readily when placed under suitable pressure, e.g. in the presence of antibiotics which bind to functionally critical regions of rRNAs. While the structures of rRNAs have been altered intentionally for decades to enable the study of their role(s) in the mechanism of peptide bond formation, it is remarkable that the purposeful alteration of rRNA structure to enable the elaboration of proteins and peptides containing non-canonical amino acids has occurred only recently. In this Perspective, we summarize the history of rRNA modifications, and demonstrate how the intentional modification of 23S rRNA in regions critical for peptide bond formation now enables the direct ribosomal incorporation of D-amino acids, β-amino acids, dipeptides and dipeptidomimetic analogues of the normal proteinogenic L-α-amino acids. While proteins containing metabolically important functional groups such as carbohydrates and phosphate groups are normally elaborated by the post-translational modification of nascent polypeptides, the use of modified ribosomes to produce such polymers directly is also discussed. Finally, we describe the elaboration of such modified proteins both in vitro and in bacterial cells, and suggest how such novel biomaterials may be exploited in future studies.

Engineered ribosomes with tethered subunits for expanding biological function

“…The extraordinarily versatile catalytic capacity of the ribosome has driven extensive efforts to harness it for novel functions, such as reprogramming the genetic code 9–13 . For example, the ability to modify the ribosome’s active site to work with substrates beyond those found in nature such as mirror-image (D-α-) and backbone-extended (β- and γ-) amino acids 14,15 , could enable the synthesis of new classes of sequence-defined polymers to meet many goals of biotechnology and medicine 11,16 . Unfortunately, cell viability constraints limit the alterations that can be made to the ribosome.…”

Emerging Methods for Efficient and Extensive Incorporation of Non-canonical Amino Acids Using Cell-Free Systems

“…Besides the normal proteinogenic amino acids, wild-type ribosome is also able to accept some analogs of L -α-amino acids ( Dedkova and Hecht, 2019 ). Nonetheless, ncAAs with modified chemical backbone such as partial D -amino acids were incompatible with the wild-type translation machinery ( Melnikov et al, 2019 ).…”

Ribosome-mediated polymerization of long chain carbon and cyclic amino acids into peptides in vitro

“…In nature, only a limited set of α- l -amino acid monomers are utilized by this system, thereby limiting the potential diversity of polymers that can be synthesized. Over the past two decades, however, efforts to expand the genetic code have shown that the natural translation system is capable of selectively incorporating a wide range of non-canonical monomers 1 – 5 . These monomers include α- 6 , β- 7 – 9 , γ- 10 – 12 , D- 13 , 14 , aromatic 15 – 17 , aliphatic 15 , 18 , malonyl 16 , N-alkylated 19 , and oligomeric amino acid analogs 10 , 20 , 21 , among others (Fig.…”