Understanding the assembly principles of biological macromolecular complexes remains a significant challenge, due to the complexity of the systems and the difficulties in developing experimental approaches. As a ribonucleoprotein complex, the ribosome serves as a model system for the profiling of macromolecular complex assembly. In this work, we report an ensemble of large ribosomal subunit intermediate structures that accumulate during synthesis in a near-physiological and co-transcriptional in vitro reconstitution system. Thirteen pre-50S intermediate maps covering the entire assembly process were resolved using cryo-EM single-particle analysis and heterogeneous subclassification. Segmentation of the set of density maps reveals that the 50S ribosome intermediates assemble based on fourteen cooperative assembly blocks, including the smallest assembly core reported to date, which is composed of a 600-nucleotide-long folded rRNA and three ribosomal proteins. The cooperative blocks assemble onto the assembly core following defined dependencies, revealing the parallel pathways at both early and late assembly stages of the 50S subunit.
We have created a bacterial semisynthetic organism (SSO)
that retains
an unnatural base pair (UBP) in its DNA, transcribes it into mRNA
and tRNA with cognate unnatural codons and anticodons, and after the
tRNA is charged with a noncanonical amino acid synthesizes proteins
containing the noncanonical amino acid. Here, we report the first
progress toward the creation of eukaryotic SSOs. After demonstrating
proof-of-concept with human HEK293 cells, we show that a variety of
different unnatural codon–anticodon pairs can efficiently mediate
the synthesis of unnatural proteins in CHO cells. Interestingly, we
find that there are both similarities and significant differences
between how the prokaryotic and eukaryotic ribosomes recognize the
UBP, with the eukaryotic ribosome appearing more tolerant. The results
represent the first progress toward eukaryotic SSOs and, in fact,
suggest that such SSOs might be able to retain more unnatural information
than their bacterial counterparts.
Previously, we evolved
a DNA polymerase, SFM4-3, for the recognition
of substrates modified at their 2′ positions with a fluoro, O-methyl, or azido substituent. Here we use SFM4-3 to synthesize
2′-azido-modified DNA; we then use the azido group to attach
different, large hydrophobic groups via click chemistry. We show that
SFM4-3 recognizes the modified templates under standard conditions,
producing natural DNA and thereby allowing amplification. To demonstrate
the utility of this remarkable property, we use SFM4-3 to select aptamers
with large hydrophobic 2′ substituents that bind human neutrophil
elastase or the blood coagulation protein factor IXa. The results
indicate that SFM4-3 should facilitate the discovery of aptamers that
adopt novel and perhaps more protein-like folds with hydrophobic cores
that in turn allow them to access novel activities.
Mass spectrometry is an important method for analysis of modified nucleosides ubiquitously present in cellular RNAs, in particular for ribosomal and transfer RNAs that play crucial roles in mRNA translation and decoding. Furthermore, modifications have effect on the lifetimes of nucleic acids in plasma and cells and are consequently incorporated into RNA therapeutics. To provide an analytical tool for sequence characterization of modified RNAs, we developed Pytheas, an open-source software package for automated analysis of tandem MS data for RNA. The main features of Pytheas are flexible handling of isotope labeling and RNA modifications, with false discovery rate statistical validation based on sequence decoys. We demonstrate bottom-up mass spectrometry characterization of diverse RNA sequences, with broad applications in the biology of stable RNAs, and quality control of RNA therapeutics and mRNA vaccines.
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