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Autonomous biogenesis of all thirty proteins of the E. coli translation machineryk

ABSTRACT: The cell-free biogenesis of the protein translation machinery is essential for the creation of a self-regenerating synthetic cell. Here, we demonstrate the autonomous and simultaneous biogenesis of all thirty proteins of the translation machinery of E. coli in a reconstituted transcription-translation system. We first established self-regeneration of every translation protein by determining the threshold concentration required for its own synthesis from a synthetic gene coding for the protein, thereby demonstrating the functionality of all nascent proteins, separately. Simultaneous biogenesis of multiple translation proteins at their threshold resulted in delayed synthesis below detection levels. To achieve self-regeneration of multiple translation proteins, we induced boundary-free compartmentalization of the reaction by immobilizing the genes on a surface at high density. The co-localization of genes, molecular machinery, messenger RNA and nascent proteins at the surface created sufficient conditions to catalyze the simultaneous self-regeneration of sub-groups up to all thirty translation proteins, as measured by total internal reflection fluorescence on the surface. Our approach provides mechanistic insight and presents a general methodology for the biogenesis of cellular machines toward autonomous synthetic systems.

INSTRUMENT(S):

ORGANISM(S): Escherichia Coli

SUBMITTER: Amir Prior

LAB HEAD: Roy H. Bar-Ziv

PROVIDER: PXD071320 | Pride | 2025-12-09

REPOSITORIES: Pride

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			Action	DRS
	20250323_213924_20032025.sne	Other
	20250323_213924_20032025_Report.log.txt	Txt
	20250323_213924_20032025_Report.params	Other
	20250323_213924_20032025_Report.setup.txt	Txt
	20250323_213924_20032025_Report.tsv	Tabular

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Project description:Understanding which factors confer specific functions in any molecular process is fundamental to understanding the system as a whole. Genetic manipulation of cellular systems often leads to the adaptation of gene expression programs, rendering detailed mechanistic insights challenging to isolate and elucidate, particularly in developmental models. The proteome constitutes the ultimate manifestation of gene expression programs; thus, multiple layers of regulation exist to ensure the faithful execution of such programs. While current high-throughput techniques aimed at investigating regulation at the level of translation, such as ribosome profiling and nascent proteomics, can capture nuanced changes in the translational landscape, they suffer from potential biases and confounding factors imposed by adaptation of the cellular states. Cell-free translation systems have been used to elucidate molecular mechanisms for decades, but experimental setups have remained rigid and often required artificially designed mRNA constructs to probe for specialized mechanisms. Here, we detail a human tri-partite cell-free translation system based on the efficient separation of ribosomes, ribosome-depleted cytoplasmic lysate, and mRNAs, allowing for flexible reconstitution of translation reactions. In this setup, some translation components, such as the cytoplasmic lysate, can be kept constant while others, such as ribosome complexes or mRNA complements, can be varied or be subjected to various treatments. We detail several steps and conditions that allow for the translation of complete mRNA populations as input and the detection of nascent peptides from such mRNA populations using mass spectrometry. We utilized this protocol to resolve which components of the translational machinery are selectively affected by environmental and cellular stress conditions that trigger ribosome stalling and collisions, which hitherto have been unresolvable. Finally, we describe how lysates can be prepared from genetically manipulated cells and efficiently and robustly incorporated into high-throughput drug screening pipelines.

Project description:Disruptions of protein homeostasis in the endoplasmic reticulum (ER) elicit activation of the unfolded protein response (UPR), a translation- and transcription-coupled proteostatic stress response pathway. The primary translational control arm of the UPR is initiated by the PERK-dependent phosphorylation of eIF2Î±, leading to a large-scale reprogramming of translation and subsequent activation of an ATF4-mediated transcriptional program. It has remained challenging, however, to accurately evaluate the contribution of each component of the eIF2Î±/ATF4 pathway to the remodelling of transcription and translation. Here, we have used mouse embryonic fibroblasts containing either a knock-in of the non-phosphorylatable eIF2Î± S51A mutant or knock-out for ATF4 by ribosome profiling and mRNA-seq to define the specific contributions of eIF2Î± phosphoryation and ATF4 in controlling the translational and transcriptional components of the UPR. These studies show that the transcriptional and translational targets of each P-eIF2Î±, ATF4, and the other UPR gene expression programs overlapped extensively, leading to a core set of UPR genes whose robust expression in response to ER stress is driven by multiple mechanisms. The identification of other, more factor-specific targets illustrated the potential for functional specialization of the UPR. As the UPR progressed temporally, cells employed distinct combinations of transcriptional and translational mechanisms, initiated by different factors, to adapt to ongoing stress. These effects were accompanied by a buffering effect where changes in mRNA levels were coupled to opposing changes in ribosome loading, a property which makes cooperation between transcription and translation essential to confer robust protein expression. Translational analysis by ribosome profiling and mRNA-seq of PERK pathways mutants during the UPR. Mouse embryonic fibroblasts (MEFs) lacking components of the PERK pathway (eIF2a phosphorylation and ATF4) were subjected to ER stress and analyzed by ribosome profiling.

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Autonomous biogenesis of all thirty proteins of the E. coli translation machineryk

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