Translation inhibition and resource balance in the TX-TL cell-free gene expression system

Abstract Quantifying the effect of vital resources on transcription (TX) and translation (TL) helps to understand the degree to which the concentration of each resource must be regulated for achieving homeostasis. Utilizing the synthetic TX-TL system, we study the impact of nucleotide triphosphates (NTPs) and magnesium (Mg2+) on gene expression. Recent observations of the counter-intuitive phenomenon of suppression of gene expression at high NTP concentrations have led to the speculation that such suppression is due to the consumption of resources by TX, hence leaving fewer resources for TL. In this work, we investigate an alternative hypothesis: direct suppression of the TL rate via stoichiometric mismatch in necessary reagents. We observe NTP-dependent suppression even in the early phase of gene expression, contradicting the resource-limitation argument. To further decouple the contributions of TX and TL, we performed gene expression experiments with purified messenger RNA (mRNA). Simultaneously monitoring mRNA and protein abundances allowed us to extract a time-dependent translation rate. Measuring TL rates for different Mg2+ and NTP concentrations, we observe a complex resource dependence. We demonstrate that TL is the rate-limiting process that is directly inhibited by high NTP concentrations. Additional Mg2+ can partially reverse this inhibition. In several experiments, we observe two maxima of the TL rate viewed as a function of both Mg2+ and NTP concentration, which can be explained in terms of an NTP-independent effect on the ribosome complex and an NTP-Mg2+ titration effect. The non-trivial compensatory effects of abundance of different vital resources signal the presence of complex regulatory mechanisms to achieve optimal gene expression.

[1]  Takuya Ueda,et al.  Cell-free translation reconstituted with purified components , 2001, Nature Biotechnology.

[2]  N. Packard,et al.  Preparation of amino acid mixtures for cell-free expression systems , 2022 .

[3]  P. Sarnow,et al.  Initiation factor-independent translation mediated by the hepatitis C virus internal ribosome entry site. , 2006, RNA.

[4]  K. Nierhaus Mg2+, K+, and the Ribosome , 2014, Journal of bacteriology.

[5]  Vincent Noireaux,et al.  Development of an artificial cell, from self-organization to computation and self-reproduction , 2011 .

[6]  Kazufumi Hosoda,et al.  Reaction dynamics analysis of a reconstituted Escherichia coli protein translation system by computational modeling , 2017, Proceedings of the National Academy of Sciences.

[7]  R. Airas Differences in the magnesium dependences of the class I and class II aminoacyl-tRNA synthetases from Escherichia coli. , 1996, European journal of biochemistry.

[8]  I. A. Rose The state of magnesium in cells as estimated from the adenylate kinase equilibrium. , 1968, Proceedings of the National Academy of Sciences of the United States of America.

[9]  A. Kondo,et al.  ATP regulation in bioproduction , 2015, Microbial Cell Factories.

[10]  Domitilla Del Vecchio,et al.  Limitations and trade-offs in gene expression due to competition for shared cellular resources , 2014, CDC.

[11]  V. Noireaux,et al.  An E. coli cell-free expression toolbox: application to synthetic gene circuits and artificial cells. , 2012, ACS synthetic biology.

[12]  R. Murray,et al.  Gene circuit performance characterization and resource usage in a cell-free "breadboard". , 2014, ACS synthetic biology.

[13]  W. Filipowicz,et al.  The effect of magnesium-ion concentration on the translation of phage-f2 RNA in a cell-free system of Escherichia coli. , 1972, European journal of biochemistry.

[14]  Vincent Noireaux,et al.  A vesicle bioreactor as a step toward an artificial cell assembly. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[15]  Vincent Noireaux,et al.  Synthesis of 2.3 mg/ml of protein with an all Escherichia coli cell-free transcription-translation system. , 2014, Biochimie.

[16]  V. Noireaux,et al.  Preparation of amino acid mixtures for cell-free expression systems. , 2015, BioTechniques.

[17]  C. Gualerzi,et al.  Specific, efficient, and selective inhibition of prokaryotic translation initiation by a novel peptide antibiotic. , 2006, Proceedings of the National Academy of Sciences of the United States of America.

[18]  Tae-Wan Kim,et al.  Rapid production of milligram quantities of proteins in a batch cell-free protein synthesis system. , 2006, Journal of biotechnology.

[19]  G. Stan,et al.  Overloaded and stressed: whole-cell considerations for bacterial synthetic biology. , 2016, Current opinion in microbiology.

[20]  R. Bar-Ziv,et al.  Principles of cell-free genetic circuit assembly , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[21]  T. Ueda,et al.  The PURE system for the cell-free synthesis of membrane proteins , 2015, Nature Protocols.

[22]  F. Wolf,et al.  Chemistry and biochemistry of magnesium. , 2003, Molecular aspects of medicine.

[23]  M. Siemann‐Herzberg,et al.  Site-Specific Cleavage of Ribosomal RNA in Escherichia coli-Based Cell-Free Protein Synthesis Systems , 2016, PloS one.

[24]  Richard M. Murray,et al.  Protocols for Implementing an Escherichia coli Based TX-TL Cell-Free Expression System for Synthetic Biology , 2013, Journal of visualized experiments : JoVE.