Translation inhibition and resource balance in the TX-TL cell-free gene expression system
© The Author 2017. Published by Oxford University Press..
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.
| Medienart: |
E-Artikel |
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| Erscheinungsjahr: |
2017 |
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| Erschienen: |
2017 |
| Enthalten in: |
Zur Gesamtaufnahme - volume:2 |
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| Enthalten in: |
Synthetic biology (Oxford, England) - 2(2017), 1 vom: 23. Jan., Seite ysx005 |
| Sprache: |
Englisch |
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| Beteiligte Personen: |
Nagaraj, Vijayalakshmi H [VerfasserIn] |
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| Links: |
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| Themen: |
Cell-free systems |
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| Anmerkungen: |
Date Revised 02.10.2020 published: Electronic-eCollection Citation Status PubMed-not-MEDLINE |
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| doi: |
10.1093/synbio/ysx005 |
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| funding: |
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| Förderinstitution / Projekttitel: |
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| PPN (Katalog-ID): |
NLM315660767 |
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| 520 | |a © The Author 2017. Published by Oxford University Press. | ||
| 520 | |a 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 | ||
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| 700 | 1 | |a Sengupta, Anirvan M |e verfasserin |4 aut | |
| 700 | 1 | |a Sontag, Eduardo D |e verfasserin |4 aut | |
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