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Abstract The ribosome is a ribonucleoprotein complex found in all domains of life. Its role is to catalyze protein synthesis, the messenger RNA (mRNA)-templated formation of amide bonds between α-amino acid monomers. Amide bond formation occurs within a highly conserved region of the large ribosomal subunit known as the peptidyl transferase center (PTC). Here we describe the step-wise design and characterization of mini-PTC 1.1, a 284-nucleotide RNA that recapitulates many essential features of the Escherichia coli PTC. Mini-PTC 1.1 folds into a PTC-like structure under physiological conditions, even in the absence of r-proteins, and engages small molecule analogs of A- and P-site tRNAs. The sequence of mini-PTC 1.1 differs from the wild type E. coli ribosome at 12 nucleotides that were installed by a cohort of citizen scientists using the on-line video game Eterna. These base changes improve both the secondary structure and tertiary folding of mini-PTC 1.1 as well as its ability to bind small molecule substrate analogs. Here, the combined input from Eterna citizen-scientists and RNA structural analysis provides a robust workflow for the design of a minimal PTC that recapitulates many features of an intact ribosome.
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Mapping the in vivo fitness landscape of a tethered ribosome
Fitness landscapes are models of the sequence space of a genetic element that map how each sequence corresponds to its activity and can be used to guide laboratory evolution. The ribosome is a macromolecular machine that is essential for protein synthesis in all organisms. Because of the prevalence of dominant lethal mutations, a comprehensive fitness landscape of the ribosomal peptidyl transfer center (PTC) has not yet been attained. Here, we develop a method to functionally map an orthogonal tethered ribosome (oRiboT), which permits complete mutagenesis of nucleotides located in the PTC and the resulting epistatic interactions. We found that most nucleotides studied showed flexibility to mutation, and identified epistatic interactions between them, which compensate for deleterious mutations. This work provides a basis for a deeper understanding of ribosome function and malleability and could be used to inform design of engineered ribosomes with applications to synthesize next-generation biomaterials and therapeutics.
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- Award ID(s):
- 1935120
- PAR ID:
- 10504868
- Publisher / Repository:
- Science Advances
- Date Published:
- Journal Name:
- Science Advances
- Volume:
- 9
- Issue:
- 17
- ISSN:
- 2375-2548
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1126/sciadv.ade8934
