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

ABSTRACT: 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.

SUBMITTER: Jewett MC

PROVIDER: S-EPMC3964315 | biostudies-literature | 2013 Jun

REPOSITORIES: biostudies-literature

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

Jewett Michael C MC Fritz Brian R BR Timmerman Laura E LE Church George M GM

Molecular systems biology 20130625

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 condition ...[more]

PMID: 23799452

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Project description:Many mutations in genes for ribosomal proteins (r-proteins) and assembly factors cause cell stress and altered cell fate, resulting in congenital diseases collectively called ribosomopathies. Even though all such mutations depress the cell's protein synthesis capacity, they generate many different phenotypes, suggesting that the diseases are not due simply to insufficient protein synthesis capacity. To learn more, we investigated how the global transcriptome in Saccharomyces cerevisiae responds to reduced protein synthesis generated in two different ways: abolishing the assembly of new ribosomes and inhibiting ribosomal function. Our results showed that the mechanism by which protein synthesis is obstructed affects the ribosomal protein transcriptome differentially: ribosomal protein mRNA abundance increases during the abolition of ribosome formation but decreases during the inhibition of ribosome function. Interestingly, the ratio between mRNAs from some, but not all, pairs of paralogous ribosomal protein genes encoding slightly different versions of a given r-protein changed differently during the two types of stress, suggesting that expression of specific ribosomal protein paralogous mRNAs may contribute to the stress response. Unexpectedly, the abundance of transcripts for ribosome assembly factors and translation factors remained relatively unaffected by the stresses. On the other hand, the state of the translation apparatus did affect cell physiology: mRNA levels for some other proteins not directly related to the translation apparatus also changed differentially, though not coordinately with the r-protein genes, in response to the stresses. IMPORTANCE Mutations in genes for ribosomal proteins or assembly factors cause a variety of diseases called ribosomopathies. These diseases are typically ascribed to a reduction in the cell's capacity for protein synthesis. Paradoxically, ribosomal mutations result in a wide variety of disease phenotypes, even though they all reduce protein synthesis. Here, we show that the transcriptome changes differently depending on how the protein synthesis capacity is reduced. Most strikingly, inhibiting ribosome formation and ribosome function had opposite effects on the abundance of mRNA for ribosomal proteins, while genes for ribosome translation and assembly factors showed no systematic responses. Thus, the process by which the protein synthesis capacity is reduced contributes decisively to global mRNA composition. This emphasis on process is a new concept in understanding ribosomopathies and other stress responses.

Dataset Information

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

Publications

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

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