scholarly journals Cell-free translation is more variable than transcription

2016 ◽  
Author(s):  
Fabio Chizzolini ◽  
Michele Forlin ◽  
Noël Yeh Martín ◽  
Giuliano Berloffa ◽  
Dario Cecchi ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Protein Expression ◽  
Rna Polymerase ◽  
Rna Synthesis ◽  
Gc Content ◽  
Cell Extract ◽  
Pure System ◽  
Protein Levels ◽  
Transcriptional Promoters ◽  
Free Translation

ABSTRACTAlthough RNA synthesis can be reliably controlled with different T7 transcriptional promoters during cell-free gene expression with the PURE system, protein synthesis remains largely unaffected. To better control protein levels, a series of ribosome binding sites (RBS) was investigated. While RBS strength did strongly affect protein synthesis, the RBS sequence could explain less than half of the variability of the data. Protein expression was found to depend on other factors besides the strength of the RBS, including GC content. The complexity of protein synthesis in comparison to RNA synthesis was observed by the higher degree of variability associated with protein expression. This variability was also observed in an E. coli cell extract-based system. However, the coefficient of variation was larger with E. coli RNA polymerase than with T7 RNA polymerase, consistent with the increased complexity of E. coli RNA polymerase.

scholarly journals RIBONUCLEIC ACID AND PROTEIN SYNTHESIS IN MITOTIC HELA CELLS

1965 ◽  
Vol 27 (3) ◽  
pp. 565-574 ◽  
Author(s):  
Terry C. Johnson ◽  
John J. Holland
Keyword(s):  
Protein Synthesis ◽  
Rna Polymerase ◽  
Hela Cells ◽  
Messenger Rna ◽  
Rna Synthesis ◽  
Mitotic Cell ◽  
Interphase Cell ◽  
Cell Extracts ◽  
Interphase Cells ◽  
Mitotic Cells

HeLa cells arrested in mitosis were obtained in large numbers, with only very slight interphase cell contamination, by employing the agitation method of Terasima and Tolmach, and Robbins and Marcus. Protein synthesis and RNA synthesis were almost completely suppressed in mitotic cells. Active polyribosomes were nearly absent in mitotic cells as compared with interphase cells treated in the same way. Cell-free protein synthesis and RNA polymerase activity were also greatly depressed in extracts of metaphase cells. The deoxyribonucleoprotein (DNP) of condensed chromosomes from mitotic cells was less efficient as a template for Escherichia coli RNA polymerase than was DNP from interphase cells, although isolated DNA from both sources was equally active as a primer. Despite very poor endogenous amino acid incorporation by extracts of metaphase cells, polyuridylate stimulated phenylalanine incorporation by a larger factor in mitotic cell extracts than it did in interphase cell extracts. These results suggest that RNA synthesis is suppressed in mitotic cells because the condensed chromosomes cannot act as a template, and that protein synthesis is depressed at least in part because messenger RNA becomes unavailable to ribosomes. This conclusion was supported by the demonstration that cells arrested in metaphase supported multiplication of normal yields of poliovirus, thereby showing that the mitotic cell is capable of considerable synthesis of RNA and protein.

scholarly journals A Method for Cost-Effective and Rapid Characterization of Engineered T7-based Transcription Factors by Cell-Free Protein Synthesis Reveals Insights into the Regulation of T7 RNA Polymerase-Driven Expression

2019 ◽  
Author(s):  
John B. McManus ◽  
Richard M. Murray ◽  
Peter A. Emanuel ◽  
Matthew W. Lux
Keyword(s):  
Protein Synthesis ◽  
Transcription Factors ◽  
Protein Expression ◽  
Rna Polymerase ◽  
Circuit Design ◽  
Cost Effective ◽  
Gene Circuit ◽  
Free Protein ◽  
Cost Effective Method ◽  
Cell Free Protein Synthesis

AbstractThe T7 bacteriophage RNA polymerase (T7 RNAP) serves as a model for understanding RNA synthesis, as a tool for protein expression, and as an actuator for synthetic gene circuit design in bacterial cells and cell-free extract. T7 RNAP is an attractive tool for orthogonal protein expression in bacteria owing to its compact single subunit structure and orthogonal promoter specificity. Understanding the mechanisms underlying T7 RNAP regulation is important to the design of engineered T7-based transcription factors, which can be used in gene circuit design. To explore regulatory mechanisms for T7 RNAP-driven expression, we developed a rapid and cost-effective method to characterize engineered T7-based transcription factors using cell-free protein synthesis and an acoustic liquid handler. Using this method, we investigated the effects of the tetracycline operator’s proximity to the T7 promoter on the regulation of T7 RNAP-driven expression. Our results reveal a mechanism for regulation that functions by interfering with the transition of T7 RNAP from initiation to elongation and validates the use of the method described here to engineer future T7-based transcription factors.HighlightsDevelopment of a rapid and cost-effective method for screening synthetic promoters.Insights into the regulation of engineered T7-based transcription factors and T7 RNAP enzyme kinetics.Validation of this method by comparison with the T7 RNAP kinetic model.

scholarly journals Action of intact AP (apurinic/apyrimidinic) sites and AP sites associated with breaks on the transcription of T7 coliphage DNA by Escherichia coli RNA polymerase

1985 ◽  
Vol 229 (1) ◽  
pp. 173-181 ◽  
Author(s):  
P A Flamée ◽  
W G Verly
Keyword(s):  
Escherichia Coli ◽  
Protein Synthesis ◽  
Rna Polymerase ◽  
Rna Synthesis ◽  
Alkylating Agents ◽  
Experimental Conditions ◽  
E Coli ◽  
Template Strand ◽  
Ap Sites ◽  
Ap Site

The effect of apurinic/apyrimidinic (AP) sites in DNA on RNA and protein synthesis was studied in vitro using T7 coliphage DNA. Initiation of RNA synthesis by Escherichia coli RNA polymerase was synchronized and heparin was used to prevent reinitiation. When the T7 DNA contained AP sites, the rate of RNA synthesis was decreased but it remained higher than the values calculated on the assumption that an AP site in the transcribed strand is a complete block to the enzyme progression. Moreover, after the time taken by an unimpeded enzyme to go from promoter to terminator, the rate of RNA synthesis remained elevated and the number of complete RNA molecules (7000 nucleotides) continued to increase for some time. These results suggest that, if the E. coli RNA polymerase is stopped by an AP site, most often, after a pause, the enzyme resumes elongation of the RNA chain which is continuous over the AP site. Sometimes however, RNA synthesis is definitively interrupted during the pause; the probability of interruption has been estimated to be 0.3 in our experimental conditions. When a nick is placed 5′ to the AP site by an AP endonuclease, the results are similar: most often, the RNA chain is synthesized without interruption past the nick in the template strand. The pause of the E. coli RNA polymerase at this combined lesion appears to be shorter than when the AP site is intact. To investigate whether a nucleotide is placed in the RNA chain in front of the AP site in the template strand by E. coli RNA polymerase, RNA synthesis was taken to completion before using this RNA for protein synthesis and measuring the activity of gene-1 product, T7 RNA polymerase. The result suggests that, after pausing, the E. coli RNA polymerase places a nucleotide in the RNA chain when passing over an AP site. The mechanism of the delayed lethality of T7 coliphages treated with monofunctional alkylating agents, which is due to the appearance of AP sites, is discussed.

FEMALE ENDOCRINE CONTROL MECHANISMS DURING THE NEONATAL PERIOD

1972 ◽  
Vol 70 (2) ◽  
pp. 396-408 ◽  
Author(s):  
K.-D. Schulz ◽  
H. Haarmann ◽  
A. Harland
Keyword(s):  
Protein Synthesis ◽  
Reproductive System ◽  
Rna Synthesis ◽  
Neonatal Period ◽  
High Dose ◽  
Female Reproductive System ◽  
Endocrine Control ◽  
Hypothalamic Region ◽  
Rna And Protein Synthesis ◽  
Physiological Dose

ABSTRACT The present investigation deals with the oestrogen-sensitivity of the female reproductive system during the neonatal period. Newborn female guinea pigs were used as test animals. At different times after a single subcutaneous injection of a physiological dose of 0.1 μg or an unphysiologically high dose of 10 μg 17β-oestradiol/100 g body weight, the RNA- and protein-synthesis was examined in the hypothalamic region, pituitary, cerebral cortex, liver, adrenal gland, ovary and uterus. With a physiological dose an increase in organ weight, protein content, RNA-and protein-synthesis was found only in the uterus. These alterations turned out to be dose-dependent. In addition to the findings in the uterus an inhibition of the aminoacid incorporation rate occurred in the liver following the injection of the high oestradiol dose. As early as 1 hour after the administration of 0.1 μg 17β-oestradiol an almost 100% increase in uterine protein synthesis was detectable. This result demonstrates a high oestrogen-sensitivity of this organ during the neonatal period. All the other organs of the female reproductive system such as the hypothalamus, pituitary and ovary did not show any oestrogen response. Therefore the functional immaturity of the uterus during post partem life is not the result of a deficient hormone sensitivity but is correlated with the absence of a sufficient hormonal stimulus at this time. The investigation on the effects of actinomycin resulted in different reactions in the uterus and liver. In contrast to the liver a paradoxical actinomycin effect was found in the uterus after treatment with actinomycin alone. This effect is characterized by a small inhibition of RNA-synthesis and a 50% increase in protein synthesis. The treatment of the newborn test animals with actinomycin and 17β-oestradiol together abolished the oestrogen-induced stimulation of the uterine RNA-and protein-synthesis. Consequently, the effect of oestrogens during the neonatal period is also connected with the formation of new proteins via an increased DNA-directed RNA-synthesis.

scholarly journals Mechanism of SARS-CoV-2 polymerase stalling by remdesivir

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Goran Kokic ◽  
Hauke S. Hillen ◽  
Dimitry Tegunov ◽  
Christian Dienemann ◽  
Florian Seitz ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Rna Polymerase ◽  
Binding Site ◽  
Molecular Mechanism ◽  
Active Center ◽  
Rna Synthesis ◽  
Active Form ◽  
Nucleoside Triphosphate ◽  
Rna Dependent Rna Polymerase ◽  
Cryo Electron Microscopy

AbstractRemdesivir is the only FDA-approved drug for the treatment of COVID-19 patients. The active form of remdesivir acts as a nucleoside analog and inhibits the RNA-dependent RNA polymerase (RdRp) of coronaviruses including SARS-CoV-2. Remdesivir is incorporated by the RdRp into the growing RNA product and allows for addition of three more nucleotides before RNA synthesis stalls. Here we use synthetic RNA chemistry, biochemistry and cryo-electron microscopy to establish the molecular mechanism of remdesivir-induced RdRp stalling. We show that addition of the fourth nucleotide following remdesivir incorporation into the RNA product is impaired by a barrier to further RNA translocation. This translocation barrier causes retention of the RNA 3ʹ-nucleotide in the substrate-binding site of the RdRp and interferes with entry of the next nucleoside triphosphate, thereby stalling RdRp. In the structure of the remdesivir-stalled state, the 3ʹ-nucleotide of the RNA product is matched and located with the template base in the active center, and this may impair proofreading by the viral 3ʹ-exonuclease. These mechanistic insights should facilitate the quest for improved antivirals that target coronavirus replication.

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