Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

In vitro ribosome synthesis and evolution through ribosome display.

ABSTRACT: Directed evolution of the ribosome for expanded substrate incorporation and novel functions is challenging because the requirement of cell viability limits the mutations that can be made. Here we address this challenge by combining cell-free synthesis and assembly of translationally competent ribosomes with ribosome display to develop a fully in vitro methodology for ribosome synthesis and evolution (called RISE). We validate the RISE method by selecting active genotypes from a ~1.7?×?10⁷ member library of ribosomal RNA (rRNA) variants, as well as identifying mutant ribosomes resistant to the antibiotic clindamycin from a library of ~4?×?10³ rRNA variants. We further demonstrate the prevalence of positive epistasis in resistant genotypes, highlighting the importance of such interactions in selecting for new function. We anticipate that RISE will facilitate understanding of molecular translation and enable selection of ribosomes with altered properties.

SUBMITTER: Hammerling MJ

PROVIDER: S-EPMC7048773 | biostudies-literature | 2020 Feb

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

In vitro ribosome synthesis and evolution through ribosome display.

Hammerling Michael J MJ Fritz Brian R BR Yoesep Danielle J DJ Kim Do Soon DS Carlson Erik D ED Jewett Michael C MC

Nature communications 20200228 1

Directed evolution of the ribosome for expanded substrate incorporation and novel functions is challenging because the requirement of cell viability limits the mutations that can be made. Here we address this challenge by combining cell-free synthesis and assembly of translationally competent ribosomes with ribosome display to develop a fully in vitro methodology for ribosome synthesis and evolution (called RISE). We validate the RISE method by selecting active genotypes from a ~1.7 × 10<sup>7</ ...[more]

PMID: 32111839

Similar Datasets

In vitro evolution of ?-hemolysin using a liposome display.

Project description:In vitro methods have enabled the rapid and efficient evolution of proteins and successful generation of novel and highly functional proteins. However, the available methods consider only globular proteins (e.g., antibodies, enzymes) and not membrane proteins despite the biological and pharmaceutical importance of the latter. In this study, we report the development of a method called liposome display that can evolve the properties of membrane proteins entirely in vitro. This method, which involves in vitro protein synthesis inside liposomes, which are cell-sized phospholipid vesicles, was applied to the pore-forming activity of ?-hemolysin, a membrane protein derived from Staphylococcus aureus. The obtained ?-hemolysin mutant possessed only two point mutations but exhibited a 30-fold increase in its pore-forming activity compared with the WT. Given the ability to synthesize various membrane proteins and modify protein synthesis and functional screening conditions, this method will allow for the rapid and efficient evolution of a wide range of membrane proteins.

| S-EPMC3801045 | biostudies-literature

Translational control through ribosome traffic jams and aborted protein synthesis

Project description:Translational control is a widespread mode of gene regulation in organisms ranging from bacteria to mammals. Computational models posit that translational control of protein expression during elongation is exerted through a traffic jam of multiple ribosomes at ribosome pause sites on mRNAs. Yet neither the in vivo frequency of ribosome traffic jams nor the contribution of such traffic jams to protein expression has been measured in any organism. Here we show that upon starvation for single amino acids in the bacterium Escherichia coli, ribosome traffic jams are pervasive across the transcriptome, but they occur at only a subset of codons cognate to the limiting amino acid, and their severity is determined by the translation efficiency of mRNAs. Surprisingly, a computational model based on the observed traffic jams at ribosome pause sites is quantitatively inconsistent with measured protein synthesis rates. By comparison, a model incorporating abortion of protein synthesis at ribosome pause sites in addition to ribosome traffic jams predicts protein synthesis rate with higher accuracy. Consistent with the latter model, a significant fraction of the nascent polypeptides at ribosome pause sites is degraded through the activity of the transfer-messenger RNA during amino acid starvation in E. coli. Our work provides a minimal, experimentally-constrained model for predicting protein expression from ribosome dynamics, and it suggests the existence of a trade-off between the cellular translational capacity and the processivity of protein synthesis in vivo. 6 samples for ribosome profiling and 5 samples for total mRNA profiling

2014-05-15 | E-GEOD-51052 | biostudies-arrayexpress

In vitro integration of ribosomal RNA synthesis, ribosome assembly, and translation.

Project description:Purely in vitro ribosome synthesis could provide a critical step towards unraveling the systems biology of ribosome biogenesis, constructing minimal cells from defined components, and engineering ribosomes with new functions. Here, as an initial step towards this goal, we report a method for constructing Escherichia coli ribosomes in crude S150 E. coli extracts. While conventional methods for E. coli ribosome reconstitution are non-physiological, our approach attempts to mimic chemical conditions in the cytoplasm, thus permitting several biological processes to occur simultaneously. Specifically, our integrated synthesis, assembly, and translation (iSAT) technology enables one-step co-activation of rRNA transcription, assembly of transcribed rRNA with native ribosomal proteins into functional ribosomes, and synthesis of active protein by these ribosomes in the same compartment. We show that iSAT makes possible the in vitro construction of modified ribosomes by introducing a 23S rRNA mutation that mediates resistance against clindamycin. We anticipate that iSAT will aid studies of ribosome assembly and open new avenues for making ribosomes with altered properties.

| S-EPMC3964315 | biostudies-literature

TSC1 sets the rate of ribosome export and protein synthesis through nucleophosmin translation.

Project description:Nucleophosmin (B23) is a nucleolar phosphoprotein that has been implicated in numerous cellular processes. In particular, nucleophosmin interacts with nucleolar components of newly synthesized ribosomes to promote ribosome nuclear export. Nucleophosmin is a classic mitogen-induced protein, with changes in its expression correlating with growth factor stimulation. In this study, we examined the underlying mechanism of nucleophosmin induction and showed that hyperproliferative signals emanating from oncogenic H-Ras(V12) cause tremendous increases in nucleophosmin protein expression. Nucleophosmin protein accumulation was dependent on mammalian target of rapamycin (mTOR) activation, as rapamycin completely prevented nucleophosmin induction. Consistent with this finding, genetic ablation of Tsc1, a major upstream inhibitor of mTOR, resulted in nucleophosmin protein induction through increased translation of existing nucleophosmin mRNAs. Increases in nucleophosmin protein accumulation were suppressed by reintroduction of TSC1. Induction of nucleophosmin through Tsc1 loss resulted in a greater pool of actively translating ribosomes in the cytoplasm, higher overall rates of protein synthesis, and increased cell proliferation, all of which were dependent on efficient nucleophosmin nuclear export. Nucleophosmin protein accumulation in the absence of Tsc1 promoted the nuclear export of maturing ribosome subunits, providing a mechanistic link between TSC1/mTOR signaling, nucleophosmin-mediated nuclear export of ribosome subunits, protein synthesis levels, and cell growth.

| S-EPMC2859708 | biostudies-literature

Ribosome display and selection of single-chain variable fragments effectively inhibit growth and progression of microspheres in vitro and in vivo.

Project description:Distinguishing the surface markers of cancer stem cells (CSCs) is a useful method for early diagnosis and treatment of tumors, as CSCs may participate in tumorigenesis and metastasis by migrating into the circulatory system. However, the potential targets of CSCs are expressed at low levels in the natural state and are always changing. Thus, dynamic screening has been reported to be an effective measure for exploring CSC markers. In recent years, diverse single-chain variable fragments (scFvs) have been widely used in immunotherapy. In this study, we determined that the scFvs, screened using RD, had a high affinity to microspheres and could inhibit their progression. We also observed that the selected scFvs underwent evolution in vitro, and antitumor-associated proteins were successfully expressed. Combined with chemotherapy, the scFvs had a synergistic effect on the inhibition of the microspheres' progression in vitro and in vivo, which could be ascribed to their high affinity for stem-like cells and the inhibition of the microspheres' collective behaviors. In addition, proteins inhibiting CD44+ /CD24+ and MAPK were involved. Our data indicated that dynamic screening of the scFvs in a natural state was of great significance in the inhibition of the microspheres in vitro and in vivo.

| S-EPMC5980252 | biostudies-literature

Ribosome Structure, Function, and Early Evolution.

Project description:Ribosomes are among the largest and most dynamic molecular motors. The structure and dynamics of translation initiation and elongation are reviewed. Three ribosome motions have been identified for initiation and translocation. A swivel motion between the head/beak and the body of the 30S subunit was observed. A tilting dynamic of the head/beak versus the body of the 30S subunit was detected using simulations. A reversible ratcheting motion was seen between the 30S and the 50S subunits that slide relative to one another. The 30S⁻50S intersubunit contacts regulate translocation. IF2, EF-Tu, and EF-G are homologous G-protein GTPases that cycle on and off the same site on the ribosome. The ribosome, aminoacyl-tRNA synthetase (aaRS) enzymes, transfer ribonucleic acid (tRNA), and messenger ribonucleic acid (mRNA) form the core of information processing in cells and are coevolved. Surprisingly, class I and class II aaRS enzymes, with distinct and incompatible folds, are homologs. Divergence of class I and class II aaRS enzymes and coevolution of the genetic code are described by analysis of ancient archaeal species.

| S-EPMC6337491 | biostudies-literature

De novo proteins from random sequences through in vitro evolution.

Project description:Natural proteins are the result of billions of years of evolution. The earliest predecessors of today's proteins are believed to have emerged from random polypeptides. While we have no means to determine how this process exactly happened, there is great interest in understanding how it reasonably could have happened. We are reviewing how researchers have utilized in vitro selection and molecular evolution methods to investigate plausible scenarios for the emergence of early functional proteins. The studies range from analyzing general properties and structural features of unevolved random polypeptides to isolating de novo proteins with specific functions from synthetic randomized sequence libraries or generating novel proteins by combining evolution with rational design. While the results are exciting, more work is needed to fully unravel the mechanisms that seeded protein-dominated biology.

| S-EPMC8222087 | biostudies-literature

Selection of a T7 promoter mutant with enhanced in vitro activity by a novel multi-copy bead display approach for in vitro evolution.

Project description:In vitro evolution of nucleic acids and proteins is a powerful strategy to optimize their biological and physical properties. To select proteins with the desired phenotype from large gene libraries, the proteins need to be linked to the gene they are encoded by. To facilitate selection of the desired phenotype and isolation of the encoding DNA, a novel bead display approach was developed, in which each member of a library of beads is first linked to multiple copies of a clonal gene variant by emulsion polymerase chain reaction. Beads are transferred to a second emulsion for an in vitro transcription-translation reaction, in which the protein encoded by each bead's amplicon covalently binds to the bead present in the same picoliter reactor. The beads then contain multiple copies of a clonal gene variant and multiple molecules of the protein encoded by the bead's gene variant and serve as the unit of selection. As a proof of concept, we screened a randomized library of the T7 promoter for high expression levels by flow cytometry and identified a T7 promoter variant with an ~10-fold higher in vitro transcriptional activity, confirming that the multi-copy bead display approach can be efficiently applied to in vitro evolution.

| S-EPMC3592457 | biostudies-literature

The origin and evolution of the ribosome.

Project description:The origin and early evolution of the active site of the ribosome can be elucidated through an analysis of the ribosomal proteins' taxonomic block structures and their RNA interactions. Comparison between the two subunits, exploiting the detailed three-dimensional structures of the bacterial and archaeal ribosomes, is especially informative.The analysis of the differences between these two sites can be summarized as follows: 1) There is no self-folding RNA segment that defines the decoding site of the small subunit; 2) there is one self-folding RNA segment encompassing the entire peptidyl transfer center of the large subunit; 3) the protein contacts with the decoding site are made by a set of universal alignable sequence blocks of the ribosomal proteins; 4) the majority of those peptides contacting the peptidyl transfer center are made by bacterial or archaeal-specific sequence blocks.These clear distinctions between the two subunit active sites support an earlier origin for the large subunit's peptidyl transferase center (PTC) with the decoding site of the small subunit being a later addition to the ribosome. The main implications are that a single self-folding RNA, in conjunction with a few short stabilizing peptides, formed the precursor of the modern ribosomal large subunit in association with a membrane.

| S-EPMC2386862 | biostudies-literature

Evolution of the ribosome at atomic resolution.

Project description:The origins and evolution of the ribosome, 3-4 billion years ago, remain imprinted in the biochemistry of extant life and in the structure of the ribosome. Processes of ribosomal RNA (rRNA) expansion can be "observed" by comparing 3D rRNA structures of bacteria (small), yeast (medium), and metazoans (large). rRNA size correlates well with species complexity. Differences in ribosomes across species reveal that rRNA expansion segments have been added to rRNAs without perturbing the preexisting core. Here we show that rRNA growth occurs by a limited number of processes that include inserting a branch helix onto a preexisting trunk helix and elongation of a helix. rRNA expansions can leave distinctive atomic resolution fingerprints, which we call "insertion fingerprints." Observation of insertion fingerprints in the ribosomal common core allows identification of probable ancestral expansion segments. Conceptually reversing these expansions allows extrapolation backward in time to generate models of primordial ribosomes. The approach presented here provides insight to the structure of pre-last universal common ancestor rRNAs and the subsequent expansions that shaped the peptidyl transferase center and the conserved core. We infer distinct phases of ribosomal evolution through which ribosomal particles evolve, acquiring coding and translocation, and extending and elaborating the exit tunnel.

| S-EPMC4104869 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data