scholarly journals Method for Rapid Analysis of Mutant RNA Polymerase Activity on Templates Containing Unnatural Nucleotides

2021 ◽  
Vol 22 (10) ◽  
pp. 5186
Author(s):  
Tatiana Egorova ◽  
Ekaterina Shuvalova ◽  
Sabina Mukba ◽  
Alexey Shuvalov ◽  
Peter Kolosov ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
High Efficiency ◽  
High Sensitivity ◽  
High Specificity ◽  
Polymerase Activity ◽  
Rapid Analysis ◽  
Rna Polymerases ◽  
Translation System ◽  
Free System ◽  
Rna Polymerase Activity

Pairs of unnatural nucleotides are used to expand the genetic code and create artificial DNA or RNA templates. In general, an approach is used to engineer orthogonal systems capable of reading codons comprising artificial nucleotides; however, DNA and RNA polymerases capable of recognizing unnatural nucleotides are required for amplification and transcription of templates. Under favorable conditions, in the presence of modified nucleotide triphosphates, DNA polymerases are able to synthesize unnatural DNA with high efficiency; however, the currently available RNA polymerases reveal high specificity to the natural nucleotides and may not easily recognize the unnatural nucleotides. Due to the absence of simple and rapid methods for testing the activity of mutant RNA polymerases, the development of RNA polymerase recognizing unnatural nucleotides is limited. To fill this gap, we developed a method for rapid analysis of mutant RNA polymerase activity on templates containing unnatural nucleotides. Herein, we optimized a coupled cell-free translation system and tested the ability of three unnatural nucleotides to be transcribed by different T7 RNA polymerase mutants, by demonstrating high sensitivity and simplicity of the developed method. This approach can be applied to various unnatural nucleotides and can be simultaneously scaled up to determine the activity of numerous polymerases on different templates. Due to the simplicity and small amounts of material required, the developed cell-free system provides a highly scalable and versatile tool to study RNA polymerase activity.

scholarly journals Inhibition of hepatic deoxyribonucleic acid-dependent ribonucleic acid polymerases by the exotoxin of Bacillus thuringiensis in comparison with the effects of α-amanitin and cordycepin

1972 ◽  
Vol 129 (1) ◽  
pp. 153-166 ◽  
Author(s):  
Edward A. Smuckler ◽  
Asen A. Hadjiolov
Keyword(s):  
Bacillus Thuringiensis ◽  
Rna Polymerase ◽  
Competitive Inhibition ◽  
Polymerase Activity ◽  
Rna Polymerases ◽  
Similar Action ◽  
Isolated Nuclei ◽  
Nuclear Rna ◽  
Rna Polymerase Activity

The action of Bacillus thuringiensis exotoxin, a structural analogue of ATP, on mouse liver DNA-dependent RNA polymerases was studied and its effects were compared with those of α-amanitin and cordycepin. (1) Administration of exotoxin in vivo caused a marked decrease in RNA polymerase activity of isolated nuclei at various concentrations of Mg2+, Mn2+and (NH4)2SO4. A similar action was recorded after addition of exotoxin to isolated nuclei from control or exotoxin-treated mice. (2) Chromatographic separation of nuclear RNA polymerases from mice treated in vivo with exotoxin showed a drastic decrease of the peak of nucleoplasmic RNA polymerase, whereas the peak of nucleolar RNA polymerase remained unaltered. The same effect was observed after administration of α-amanitin in vivo, but cordycepin did not alter the relative amounts of the two main RNA polymerase peaks. (3) Administration of exotoxin in vivo did not alter the template activity of isolated DNA or chromatin tested with different fractions of RNA polymerase from control or exotoxin-treated mice. (4) Addition of exotoxin to isolated liver RNA polymerases inhibited both enzyme fractions. However, the α-amanitin-sensitive RNA polymerase was also 50–100-fold more sensitive to exotoxin inhibition than was the α-amanitin-insensitive RNA polymerase. Kinetic analysis indicated the exotoxin produces a competitive inhibition with ATP on the nucleolar enzyme, but a mixed type of inhibition with nucleoplasmic enzyme. The results obtained indicate that the B. thuringiensis exotoxin inhibits liver RNA synthesis by affecting nuclear RNA polymerases, showing a preferential inhibition of the nucleoplasmic α-amanitin-sensitive RNA polymerase.

Effects of Phospholipases on RNA Polymerase Activity in Preparations from Rat Brain

1971 ◽  
Vol 49 (12) ◽  
pp. 1318-1325 ◽  
Author(s):  
I. A. Menon
Keyword(s):  
Rna Polymerase ◽  
Rat Brain ◽  
Phospholipase C ◽  
Ammonium Sulfate ◽  
Enzyme Preparation ◽  
Brain Mitochondria ◽  
Polymerase Activity ◽  
Rna Polymerases ◽  
Phospholipase A ◽  
Rna Polymerase Activity

Phospholipase A and phospholipase C increased the RNA polymerase activities in presence of Mg2+ or Mn2+ of aggregate enzyme preparation from rat brain. The stimulatory effects were not observed when the RNA polymerase activities were determined in presence of 0.4 M ammonium sulfate, KCl, or NaCl. Trypsin, pronase, and papain were found to enhance the RNA polymerase activity in presence of Mn2+ whereas they had little or no effect on the activity in presence of Mg2+. The phospholipase had relatively less effect on the RNA polymerase activity of frozen and thawed or sonicated aggregate enzyme preparation. Treatment with phospholipase A solubilized approximately 50% of the RNA polymerase. The RNA polymerase activity of brain mitochondria was stimulated by treatment with the phospholipases. The results indicate that phospholipids associated with chromatin and RNA polymerase, as well as those in mitochondria, influence the activity of the RNA polymerases.

scholarly journals Differential inhibition of mammalian ribonucleic acid polymerases by an exotoxin from Bacillus thuringiensis. The direct observation of nucleoplasmic ribonucleic acid polymerase activity in intact nuclei

1972 ◽  
Vol 127 (4) ◽  
pp. 619-624 ◽  
Author(s):  
T. Beebee ◽  
A. Korner ◽  
R. P. M. Bond
Keyword(s):  
Ionic Strength ◽  
Bacillus Thuringiensis ◽  
Rna Polymerase ◽  
Ribonucleic Acid ◽  
Polymerase Activity ◽  
High Ionic Strength ◽  
Rna Polymerases ◽  
Differential Inhibition ◽  
Rna Polymerase Activity ◽  
Soluble Enzymes

The effects of the exotoxin from Bacillus thuringiensis on DNA-dependent RNA polymerases from rat liver were examined. The exotoxin inhibits all RNA polymerase activity at both low and high ionic strength in intact nuclei, and soluble enzymes are similarly affected. This inhibition is relieved by ATP. Dephosphorylated exotoxin did not inhibit the soluble enzymes. Nucleolar and nucleoplasmic RNA polymerases respond to different concentration ranges of exotoxin, and the compound can be used in intact nuclei to isolate the nucleoplasmic activity.

Pharmaceutical interference of the EWS-FLI1-driven transcriptome by co-targeting H3K27ac and RNA polymerase activity in Ewing Sarcoma

2021 ◽  
pp. molcanther.MCT-20-0489-A.2020
Author(s):  
Daniel A. R. Heisey ◽  
Sheeba Jacob ◽  
Timonthy L Lochmann ◽  
Richard Kurupi ◽  
Maninderjit S. Ghotra ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Ewing Sarcoma ◽  
Polymerase Activity ◽  
Rna Polymerase Activity

RNA polymerase activity in virions from Ustilago maydis

1984 ◽  
Vol 4 (1) ◽  
pp. 188-194
Author(s):  
B S Ben-Tzvi ◽  
Y Koltin ◽  
M Mevarech ◽  
A Tamarkin
Keyword(s):  
Rna Polymerase ◽  
Viral Genome ◽  
Ustilago Maydis ◽  
Polymerase Activity ◽  
Reaction Products ◽  
Double Stranded Rna ◽  
Rna Molecules ◽  
Rna Transcripts ◽  
Rna Polymerase Activity

RNA polymerase activity is associated with the double-stranded RNA virions of Ustilago maydis. The reaction products of the polymerase activity are single-stranded RNA molecules. The RNA molecules synthesized are homologous to the three classes of double-stranded RNA molecules that typify the viral genome. The single-stranded RNA synthesized is released from the virions. The molecular weight of the single-stranded RNA transcripts is about half the size of the double-stranded RNA segments, and thus, it appears that in the in vitro reaction, full-length transcripts can be obtained.

scholarly journals HDAC6 Restricts Influenza A Virus by Deacetylation of the RNA Polymerase PA Subunit

2018 ◽  
Vol 93 (4) ◽  
Author(s):  
Huan Chen ◽  
Yingjuan Qian ◽  
Xin Chen ◽  
Zhiyang Ruan ◽  
Yuetian Ye ◽  
...  
Keyword(s):  
Life Cycle ◽  
Rna Polymerase ◽  
Influenza A Virus ◽  
Influenza A ◽  
Molecular Mechanisms ◽  
Polymerase Activity ◽  
Negative Regulator ◽  
Host Protein ◽  
Spectrometric Analysis ◽  
Rna Polymerase Activity

ABSTRACT The life cycle of influenza A virus (IAV) is modulated by various cellular host factors. Although previous studies indicated that IAV infection is controlled by HDAC6, the deacetylase involved in the regulation of PA remained unknown. Here, we demonstrate that HDAC6 acts as a negative regulator of IAV infection by destabilizing PA. HDAC6 binds to and deacetylates PA, thereby promoting the proteasomal degradation of PA. Based on mass spectrometric analysis, Lys(664) of PA can be deacetylated by HDAC6, and the residue is crucial for PA protein stability. The deacetylase activity of HDAC6 is required for anti-IAV activity, because IAV infection was enhanced due to elevated IAV RNA polymerase activity upon HDAC6 depletion and an HDAC6 deacetylase dead mutant (HDAC6-DM; H216A, H611A). Finally, we also demonstrate that overexpression of HDAC6 suppresses IAV RNA polymerase activity, but HDAC6-DM does not. Taken together, our findings provide initial evidence that HDAC6 plays a negative role in IAV RNA polymerase activity by deacetylating PA and thus restricts IAV RNA transcription and replication. IMPORTANCE Influenza A virus (IAV) continues to threaten global public health due to drug resistance and the emergence of frequently mutated strains. Thus, it is critical to find new strategies to control IAV infection. Here, we discover one host protein, HDAC6, that can inhibit viral RNA polymerase activity by deacetylating PA and thus suppresses virus RNA replication and transcription. Previously, it was reported that IAV can utilize the HDAC6-dependent aggresome formation mechanism to promote virus uncoating, but HDAC6-mediated deacetylation of α-tubulin inhibits viral protein trafficking at late stages of the virus life cycle. These findings together will contribute to a better understanding of the role of HDAC6 in regulating IAV infection. Understanding the molecular mechanisms of HDAC6 at various periods of viral infection may illuminate novel strategies for developing antiviral drugs.

RNA polymerase activity in germinating onion seeds

1972 ◽  
Vol 11 (11) ◽  
pp. 3105-3110 ◽  
Author(s):  
J.F. Payne ◽  
Arya K. Bal
Keyword(s):  
Rna Polymerase ◽  
Polymerase Activity ◽  
Rna Polymerase Activity
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