scholarly journals Translation Inhibition and Resource Balance in the Cell-Free Gene Expression System

2017 ◽  
Author(s):  
Vijayalakshmi H. Nagaraj ◽  
James M. Greene ◽  
Anirvan M. Sengupta ◽  
Eduardo D. Sontag
Keyword(s):  
Gene Expression ◽  
Resource Dependence ◽  
Alternative Hypothesis ◽  
Expression System ◽  
Resource Limitation ◽  
Independent Effect ◽  
Translation Rate ◽  
Free Gene ◽  
Suppression Of Gene Expression ◽  
The Impact

AbstractQuantifying the effect of vital resources on transcription and translation helps to understand the degree to which the concentration of each resource must be regulated for achieving homeostasis. Utilizing the synthetic transcription-translation (TX-TL) system, we study the impact of nucleotide triphosphates (NTPs) and magnesium (Mg2+), on gene expression. Recent observations of the counterintuitive 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 transcription, hence leaving fewer resources for translation. In this work, we investigate an alternative hypothesis: direct suppression of the translation 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 transcription and translation, we performed gene expression experiments with purified mRNA. Simultaneously monitoring mRNA and protein abundances allowed us to extract a time-dependent translation rate. Measuring translation rates for different Mg2+ and NTP concentrations, we observe a complex resource dependence. We demonstrate that translation 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 translation 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 signals the presence of complex regulatory mechanisms to achieve optimal gene expression.

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

2017 ◽  
Vol 2 (1) ◽  
Author(s):  
Vijayalakshmi H Nagaraj ◽  
James M Greene ◽  
Anirvan M Sengupta ◽  
Eduardo D Sontag
Keyword(s):  
Gene Expression ◽  
Messenger Rna ◽  
Resource Dependence ◽  
Alternative Hypothesis ◽  
Expression System ◽  
Resource Limitation ◽  
Independent Effect ◽  
Translation Rate ◽  
Suppression Of Gene Expression ◽  
The Impact

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.

Optimized Assembly of a Multifunctional RNA-Protein Nanostructure in a Cell-Free Gene Expression System

Nano Letters ◽  
2018 ◽  
Vol 18 (4) ◽  
pp. 2650-2657 ◽  
Author(s):  
Matthaeus Schwarz-Schilling ◽  
Aurore Dupin ◽  
Fabio Chizzolini ◽  
Swati Krishnan ◽  
Sheref S. Mansy ◽  
...  
Keyword(s):  
Gene Expression ◽  
Expression System ◽  
Gene Expression System ◽  
A Cell ◽  
Free Gene

scholarly journals Deconstructing cell-free extract preparation forin vitroactivation of transcriptional genetic circuitry

2018 ◽  
Author(s):  
Adam D. Silverman ◽  
Nancy Kelley-Loughnane ◽  
Julius B. Lucks ◽  
Michael C. Jewett
Keyword(s):  
Gene Expression ◽  
Protein Synthesis ◽  
Genetic Circuits ◽  
Translation Rate ◽  
Bacterial Transcription ◽  
Crude Extracts ◽  
Run Off ◽  
Free Gene ◽  
Transcriptional Machinery

AbstractRecent advances in cell-free gene expression (CFE) systems have enabled their use for a host of synthetic biology applications, particularly for rapid prototyping of genetic circuits designed as biosensors. Despite the proliferation of cell-free protein synthesis platforms, the large number of currently existing protocols for making CFE extracts muddles the collective understanding of how the method by which an extract is prepared affects its functionality. Specifically, a key goal toward developing cell-free biosensors based on native genetic regulators is activating the transcriptional machinery present in bacterial extracts for protein synthesis. However, protein yields from genes transcribedin vitroby the nativeEscherichia coliRNA polymerase are quite low in conventional crude extracts originally optimized for expression by the bacteriophage transcriptional machinery. Here, we show that cell-free expression of genes under bacterial σ70promoters is constrained by the rate of transcription in crude extracts and that processing the extract with a ribosomal run-off reaction and subsequent dialysis can alleviate this constraint. Surprisingly, these processing steps only enhance protein synthesis in genes under native regulation, indicating that the translation rate is unaffected. We further investigate the role of other common process variants on extract performance and demonstrate that bacterial transcription is inhibited by including glucose in the growth culture, but is unaffected by flash-freezing the cell pellet prior to lysis. Our final streamlined protocol for preparing extract by sonication generates extract that facilitates expression from a diverse set of sensing modalities including protein and RNA regulators. We anticipate that this work will clarify the methodology for generating CFE extracts that are active for biosensing and will encourage the further proliferation of cell-free gene expression technology for new applications.

scholarly journals Studying the Function of Phytoplasma Effector Proteins Using a Chemical-Inducible Expression System in Transgenic Plants

2021 ◽  
Vol 22 (24) ◽  
pp. 13582
Author(s):  
Keziah M. Omenge ◽  
Florian Rümpler ◽  
Subha Suvetha Kathalingam ◽  
Alexandra C. U. Furch ◽  
Günter Theißen
Keyword(s):  
Gene Expression ◽  
Laser Scanning ◽  
Expression System ◽  
Inducible Expression ◽  
Effector Proteins ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Protein Accumulation ◽  
Disease Phenotypes ◽  
The Impact

Phytoplasmas are bacterial pathogens that live mainly in the phloem of their plant hosts. They dramatically manipulate plant development by secreting effector proteins that target developmental proteins of their hosts. Traditionally, the effects of individual effector proteins have been studied by ectopic overexpression using strong, ubiquitously active promoters in transgenic model plants. However, the impact of phytoplasma infection on the host plants depends on the intensity and timing of infection with respect to the developmental stage of the host. To facilitate investigations addressing the timing of effector protein activity, we have established chemical-inducible expression systems for the three most well-characterized phytoplasma effector proteins, SECRETED ASTER YELLOWS WITCHES’ BROOM PROTEIN 11 (SAP11), SAP54 and TENGU in transgenic Arabidopsis thaliana. We induced gene expression either continuously, or at germination stage, seedling stage, or flowering stage. mRNA expression was determined by quantitative reverse transcription PCR, protein accumulation by confocal laser scanning microscopy of GFP fusion proteins. Our data reveal tight regulation of effector gene expression and strong upregulation after induction. Phenotypic analyses showed differences in disease phenotypes depending on the timing of induction. Comparative phenotype analysis revealed so far unreported similarities in disease phenotypes, with all three effector proteins interfering with flower development and shoot branching, indicating a surprising functional redundancy of SAP54, SAP11 and TENGU. However, subtle but mechanistically important differences were also observed, especially affecting the branching pattern of the plants.

scholarly journals Correction to Optimized Assembly of a Multifunctional RNA-Protein Nanostructure in a Cell-Free Gene Expression System

Nano Letters ◽  
2019 ◽  
Vol 19 (7) ◽  
pp. 4812-4812
Author(s):  
Matthaeus Schwarz-Schilling ◽  
Aurore Dupin ◽  
Fabio Chizzolini ◽  
Swati Krishnan ◽  
Sheref S. Mansy ◽  
...  
Keyword(s):  
Gene Expression ◽  
Expression System ◽  
Gene Expression System ◽  
A Cell ◽  
Free Gene

scholarly journals Heavy Water Reduces GFP Expression in Prokaryotic Cell-Free Assays at the Translation Level While Stimulating Its Transcription

2013 ◽  
Vol 2013 ◽  
pp. 1-9 ◽  
Author(s):  
Luisa S. Hohlefelder ◽  
Tobias Stögbauer ◽  
Madeleine Opitz ◽  
Thomas M. Bayerl ◽  
Joachim O. Rädler
Keyword(s):  
Gene Expression ◽  
Heavy Water ◽  
Expression System ◽  
Inhibitory Effect ◽  
Dependent Manner ◽  
Gfp Expression ◽  
Antiproliferative Agent ◽  
Underlying Mechanisms ◽  
The Impact

Thein vitroproliferation of prokaryotic and eukaryotic cells is remarkably hampered in the presence of heavy water (D2O). Impairment of gene expression at the transcription or translation level can be the base for this effect. However, insights into the underlying mechanisms are lacking. Here, we employ a cell-free expression system for the quantitative analysis of the effect of increasing percentages of D2O on the kinetics ofin-vitroGFP expression. Experiments are designed to discriminate the rates of transcription, translation, and protein folding using pDNA and mRNA vectors, respectively. We find that D2O significantly stimulates GFP expression at the transcription level but acts as a suppressor at translation and maturation (folding) in a linear dose-dependent manner. At a D2O concentration of 60%, the GFP expression rate was reduced to 40% of an undisturbed sample. We observed a similar inhibition of GFP expression by D2O in a recombinantEscherichia colistrain, although the inhibitory effect is less pronounced. These results demonstrate the suitability of cell-free systems for quantifying the impact of heavy water on gene expression and establish a platform to further assess the potential therapeutic use of heavy water as antiproliferative agent.

scholarly journals In silico Design of Linear DNA for Robust Cell-Free Gene Expression

2021 ◽  
Vol 9 ◽  
Author(s):  
Xinjie Chen ◽  
Yuan Lu
Keyword(s):  
Gene Expression ◽  
Protein Expression ◽  
High Throughput Screening ◽  
Expression System ◽  
Dna Templates ◽  
Linear Dna ◽  
Low Protein ◽  
Cell Extracts ◽  
Free Gene ◽  
Expression Templates

Cell-free gene expression systems with linear DNA expression templates (LDETs) have been widely applied in artificial cells, biochips, and high-throughput screening. However, due to the degradation caused by native nucleases in cell extracts, the transcription with linear DNA templates is weak, thereby resulting in low protein expression level, which greatly limits the development of cell-free systems using linear DNA templates. In this study, the protective sequences for stabilizing linear DNA and the transcribed mRNAs were rationally designed according to nucleases’ action mechanism, whose effectiveness was evaluated through computer simulation and cell-free gene expression. The cell-free experiment results indicated that, with the combined protection of designed sequence and GamS protein, the protein expression of LDET-based cell-free systems could reach the same level as plasmid-based cell-free systems. This study would potentially promote the development of the LDET-based cell-free gene expression system for broader applications.

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