Ribo-T is a ribosome with covalently tethered subunits where core 16S and 23S ribosomal RNAs form a single chimeric molecule. Ribo-T makes possible a functionally orthogonal ribosome–mRNA system in cells. Unfortunately, use of Ribo-T has been limited because of low activity of its original version. Here, to overcome this limitation, we use an evolutionary approach to select new tether designs that are capable of supporting faster cell growth and increased protein expression. Further, we evolve new orthogonal Ribo-T/mRNA pairs that function in parallel with, but independent of, natural ribosomes and mRNAs, increasing the efficiency of orthogonal protein expression. The Ribo-T with optimized designs is able to synthesize a diverse set of proteins, and can also incorporate multiple non-canonical amino acids into synthesized polypeptides. The enhanced Ribo-T designs should be useful for exploring poorly understood functions of the ribosome and engineering ribosomes with altered catalytic properties.
Ribosome engineering is a powerful approach for expanding the catalytic potential of the protein synthesis apparatus. Due to the potential detriment the properties of the engineered ribosome may have on the cell, the designer ribosome needs to be functionally isolated from the translation machinery synthesizing cellular proteins. One solution to this problem was offered by Ribo-T, an engineered ribosome with tethered subunits which, while producing a desired protein, could be excluded from general translation. Here, we provide a conceptually different design of a cell with two orthogonal protein synthesis systems, where Ribo-T produces the proteome, while the dissociable ribosome is committed to the translation of a specific mRNA. The utility of this system is illustrated by generating a comprehensive collection of mutants with alterations at every rRNA nucleotide of the peptidyl transferase center and isolating gain-of-function variants that enable the ribosome to overcome the translation termination blockage imposed by an arrest peptide.
more » « less- Award ID(s):
- 1716766
- NSF-PAR ID:
- 10154083
- Publisher / Repository:
- Nature Publishing Group
- Date Published:
- Journal Name:
- Nature Communications
- Volume:
- 11
- Issue:
- 1
- ISSN:
- 2041-1723
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Abstract No method exists to measure large-scale translation of genes in uncultured organisms in microbiomes. To overcome this limitation, we develop MetaRibo-Seq, a method for simultaneous ribosome profiling of tens to hundreds of organisms in microbiome samples. MetaRibo-Seq was benchmarked against gold-standard Ribo-Seq in a mock microbial community and applied to five different human fecal samples. Unlike RNA-Seq, Ribo-Seq signal of a predicted gene suggests it encodes a translated protein. We demonstrate two applications of this technique: First, MetaRibo-Seq identifies small genes, whose identification until now has been challenging. For example, MetaRibo-Seq identifies 2,091 translated, previously unannotated small protein families from five fecal samples, more than doubling the number of small proteins predicted to exist in this niche. Second, the combined application of RNA-Seq and MetaRibo-Seq identifies differences in the translation of transcripts. In summary, MetaRibo-Seq enables comprehensive translational profiling in microbiomes and identifies previously unannotated small proteins.
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Abstract A crucial step in functional genomics is identifying actively translated open reading frames (ORFs) and linking them to biological functions. The challenge lies in identifying short ORFs, as their identification is greatly influenced by data quality and depth. Here, we improved the coverage of super-resolution Ribo-seq in Arabidopsis (Arabidopsis thaliana), revealing uncharacterized translation events for nuclear, chloroplastic, and mitochondrial genes. Assisted by a transcriptome assembly, we identified 7,751 unconventional translation events, comprising 6,996 upstream ORFs (uORFs) and 209 downstream ORFs on annotated protein-coding genes, as well as 546 ORFs in presumed non-coding RNAs. Proteomics data confirmed the production of stable proteins from some of these unannotated translation events. We present evidence of active translation from primary transcripts of tasiRNAs (TAS1–4) and microRNAs (pri-MIR163, pri-MIR169), and periodic ribosome stalling supporting co-translational decay. Additionally, we developed a method for identifying extremely short uORFs, including 370 minimum uORFs (AUG-stop), and 2,921 tiny uORFs (2–10 amino acids), and 681 uORFs that overlap with each other. Remarkably, these short uORFs exhibit strong translational repression as do longer uORFs. We also systematically discovered 594 uORFs regulated by alternative splicing, suggesting widespread isoform-specific translational control. Finally, these prevalent uORFs are associated with numerous important pathways. In summary, our improved Arabidopsis translational landscape provides valuable resources to study gene expression regulation.
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null (Ed.)Abstract Ribosome profiling, also known as Ribo-seq, has become a popular approach to investigate regulatory mechanisms of translation in a wide variety of biological contexts. Ribo-seq not only provides a measurement of translation efficiency based on the relative abundance of ribosomes bound to transcripts, but also has the capacity to reveal dynamic and local regulation at different stages of translation based on positional information of footprints across individual transcripts. While many computational tools exist for the analysis of Ribo-seq data, no method is currently available for rigorous testing of the pattern differences in ribosome footprints. In this work, we develop a novel approach together with an R package, RiboDiPA, for Differential Pattern Analysis of Ribo-seq data. RiboDiPA allows for quick identification of genes with statistically significant differences in ribosome occupancy patterns for model organisms ranging from yeast to mammals. We show that differential pattern analysis reveals information that is distinct and complimentary to existing methods that focus on translational efficiency analysis. Using both simulated Ribo-seq footprint data and three benchmark data sets, we illustrate that RiboDiPA can uncover meaningful pattern differences across multiple biological conditions on a global scale, and pinpoint characteristic ribosome occupancy patterns at single codon resolution.more » « less
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Summary The expression of chloroplast genes relies on a host of nucleus‐encoded proteins. Identification of such proteins and elucidation of their functions are ongoing challenges. We used ribosome profiling to revisit the function of the pentatricopeptide repeat protein LPE1, reported to stimulate translation of the chloroplast
psbA mRNA in Arabidopsis. Mutation of the maizeLPE1 ortholog causes a photosystem II (PSII) deficiency and a defect in translation of the chloroplastpsbJ open reading frame (ORF) but has no effect onpsbA expression. To reflect this function, we named the maize LPE1 orthologT ranslation ofp sb J lpe1 mutants likewise exhibit a loss ofpsbJ translation, and have, in addition, a decrease inpsbN translation. We detected a small decrease in ribosome occupancy on thepsbA mRNA in Arabidopsislpe1 mutants, but ribosome profiling analyses of other PSII mutants (hcf107 andhcf173 ) in conjunction within vitro RNA binding data strongly suggest that this is a secondary effect of their PSII deficiency. We conclude that maize TPJ1 promotes PSII synthesis by activating translation of thepsbJ ORF, that this function is conserved in Arabidopsis LPE1, and that an additional role for LPE1 inpsbN translation contributes to the PSII deficiency inlpe1 mutants.
