scholarly journals Transcribing Genes the Hard Way: In Vitro Reconstitution of Nanoarchaeal RNA Polymerase Reveals Unusual Active Site Properties

2021 ◽  
Vol 8 ◽  
Author(s):  
Sven Nottebaum ◽  
Robert O. J. Weinzierl
Keyword(s):  
Rna Polymerase ◽  
High Throughput Screening ◽  
Sparse Matrix ◽  
Screening Method ◽  
Fluoride Ions ◽  
Stringent Requirement ◽  
In Vitro Reconstitution ◽  
Nanoarchaeum Equitans ◽  
Complete Set

Nanoarchaea represent a highly diverged archaeal phylum that displays many unusual biological features. The Nanoarchaeum equitans genome encodes a complete set of RNA polymerase (RNAP) subunits and basal factors. Several of the standard motifs in the active center contain radical substitutions that are normally expected to render the polymerase catalytically inactive. Here we show that, despite these unusual features, a RNAP reconstituted from recombinant Nanoarchaeum subunits is transcriptionally active. Using a sparse-matrix high-throughput screening method we identified an atypical stringent requirement for fluoride ions to maximize its activity under in vitro transcription conditions.

scholarly journals A high-throughput screening method for evolving a demethylase enzyme with improved and new functionalities

2020 ◽  
Author(s):  
Yuru Wang ◽  
Christopher D Katanski ◽  
Christopher Watkins ◽  
Jessica N Pan ◽  
Qing Dai ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Screening Method ◽  
Targeted Mutagenesis ◽  
Rna Repair ◽  
Dna And Rna ◽  
Modified Dna ◽  
Dna Substrates ◽  
Trna Sequencing

Abstract AlkB is a DNA/RNA repair enzyme that removes base alkylations such as N1-methyladenosine (m1A) or N3-methylcytosine (m3C) from DNA and RNA. The AlkB enzyme has been used as a critical tool to facilitate tRNA sequencing and identification of mRNA modifications. As a tool, AlkB mutants with better reactivity and new functionalities are highly desired; however, previous identification of such AlkB mutants was based on the classical approach of targeted mutagenesis. Here, we introduce a high-throughput screening method to evaluate libraries of AlkB variants for demethylation activity on RNA and DNA substrates. This method is based on a fluorogenic RNA aptamer with an internal modified RNA/DNA residue which can block reverse transcription or introduce mutations leading to loss of fluorescence inherent in the cDNA product. Demethylation by an AlkB variant eliminates the blockage or mutation thereby restores the fluorescence signals. We applied our screening method to sites D135 and R210 in the Escherichia coli AlkB protein and identified a variant with improved activity beyond a previously known hyperactive mutant toward N1-methylguanosine (m1G) in RNA. We also applied our method to O6-methylguanosine (O6mG) modified DNA substrates and identified candidate AlkB variants with demethylating activity. Our study provides a high-throughput screening method for in vitro evolution of any demethylase enzyme.

scholarly journals Revisiting T7 RNA polymerase transcription in vitro with the Broccoli RNA aptamer as a simplified real-time fluorescent reporter

2020 ◽  
pp. jbc.RA120.014553
Author(s):  
Zachary J Kartje ◽  
Helen I Janis ◽  
Shaoni Mukhopadhyay ◽  
Keith T Gagnon
Keyword(s):  
Rna Polymerase ◽  
Real Time ◽  
High Throughput Screening ◽  
In Vitro Transcription ◽  
T7 Rna Polymerase ◽  
Rna Aptamer ◽  
Reaction Conditions ◽  
Fluorescent Reporter ◽  
Transcription In Vitro

Methods for rapid and high-throughput screening of transcription in vitro to examine reaction conditions, enzyme mutants, promoter variants, and small molecule modulators can be extremely valuable tools. However, these techniques may be difficult to establish or inaccessible to many researchers. To develop a straightforward and cost-effective platform for assessing transcription in vitro, we used the “Broccoli” RNA aptamer as a direct, real-time fluorescent transcript readout. To demonstrate the utility of our approach, we screened the effect of common reaction conditions and components on bacteriophage T7 RNA polymerase (RNAP) activity using a common quantitative PCR instrument for fluorescence detection. Several essential conditions for in vitro transcription by T7 RNAP were confirmed with this assay, including the importance of enzyme and substrate concentrations, co-variation of magnesium and nucleoside triphosphates, and the effects of several typical additives. When we used this method to assess all possible point mutants of a canonical T7 RNAP promoter, our results coincided well with previous reports. This approach should translate well to a broad variety of bacteriophage in vitro transcription systems and provides a platform for developing fluorescence-based readouts of more complex transcription systems in vitro.

scholarly journals Diversity in the signals required for nuclear accumulation of U snRNPs and variety in the pathways of nuclear transport.

1991 ◽  
Vol 113 (4) ◽  
pp. 705-714 ◽  
Author(s):  
U Fischer ◽  
E Darzynkiewicz ◽  
S M Tahara ◽  
N A Dathan ◽  
R Lührmann ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Rna Polymerase Iii ◽  
Nuclear Accumulation ◽  
Nuclear Targeting ◽  
Stringent Requirement ◽  
Pol Ii ◽  
Nuclear Localization Signals ◽  
U6 Snrna

The requirements for nuclear targeting of a number of U snRNAs have been studied by analyzing the behavior of in vitro-generated transcripts after microinjection into the cytoplasm of Xenopus oocytes. Like the previously studied U1 snRNA, U2 snRNA is excluded from the nucleus when it does not have the 2,2,7mGpppN cap structure typical of the RNA polymerase II (pol II)-transcribed U snRNAs. Surprisingly, two other pol II-transcribed U snRNAs, U4 and U5, have a much less stringent requirement for the trimethyl cap structure. The gamma-monomethyl triphosphate cap structure of the RNA polymerase III-transcribed U6 snRNA, on the other hand, is shown not to play a role in nuclear targeting. Wheat germ agglutinin, which is known to prevent the import of many proteins into the nucleus, inhibits nuclear uptake of U6, but not of U1 or U5 snRNAs. Conversely, a 2,2,7mGpppG dinucleotide analogue of the trimethyl cap structure inhibits transport of the pol II U snRNAs, but does not detectably affect the transport of either U6 snRNA or a karyophilic protein. From these results it can be deduced that U6 enters the nucleus by a pathway similar or identical to that used by karyophilic proteins. The composite nuclear localization signals of the trimethyl cap-containing U snRNPs, however, do not function in the same way as previously defined nuclear targeting signals.

scholarly journals A New High-Throughput Screening Method to Detect Antimicrobial Volatiles from Metagenomic Clone Libraries

Antibiotics ◽  
2020 ◽  
Vol 9 (11) ◽  
pp. 726
Author(s):  
Franz Stocker ◽  
Melanie M. Obermeier ◽  
Katharina Resch ◽  
Gabriele Berg ◽  
Christina A. Müller Bogotá
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Screening Method ◽  
Fusarium Culmorum ◽  
Metagenomic Library ◽  
Clone Libraries ◽  
Microbial Volatile Organic Compounds ◽  
Pure Compounds ◽  
Vitro Effect

The ever-growing spread of resistance in medicine and agriculture highlights the need to identify new antimicrobials. Microbial volatile organic compounds (VOCs) are one of the most promising groups of chemicals to meet this need. These rarely exploited molecules exhibit antimicrobial activity and their high vapour pressure makes them ideal for application in surface sterilisation, and in particular, in biofumigation. Therefore, we adapted the previously developed Two Clamp VOCs Assay (TCVA) to a new high-throughput screening for the detection of novel antifungal VOCs from metagenomic clone libraries. As a proof of concept, we tested the new high-throughput TCVA (htTCVA) by sourcing a moss metagenomic library against Fusarium culmorum. This led to the identification of five clones that inhibited the growth of mycelium and spores in vitro by up to 8% and 30% and subsequently, to the identification of VOCs that are potentially, and in part responsible for the clones’ antifungal activity. For these VOCs, the in vitro effect of the pure compounds was as high as 100%. These results demonstrate the robustness and feasibility of the htTCVA, which provides access to completely new and unexplored biosynthetic pathways and their secondary metabolites.

scholarly journals Role of the RNA Polymerase α Subunits in MetR-Dependent Activation of metE and metH: Important Residues in the C-Terminal Domain and Orientation Requirements within RNA Polymerase

2000 ◽  
Vol 182 (19) ◽  
pp. 5539-5550 ◽  
Author(s):  
Paula S. Fritsch ◽  
Mark L. Urbanowski ◽  
George V. Stauffer
Keyword(s):  
Rna Polymerase ◽  
Random Mutagenesis ◽  
Complex Surface ◽  
Α Subunit ◽  
C Terminus ◽  
Distinct Cluster ◽  
In Vitro Reconstitution ◽  
The Difference ◽  
Α Subunits

ABSTRACT Many transcription factors activate by directly interacting with RNA polymerase (RNAP). The C terminus of the RNAP α subunit (αCTD) is a common target of activators. We used both random mutagenesis and alanine scanning to identify αCTD residues that are crucial for MetR-dependent activation of metE and metH. We found that these residues localize to two distinct faces of the αCTD. The first is a complex surface consisting of residues important for α-DNA interactions, activation of both genes (residues 263, 293, and 320), and activation of either metE only (residues 260, 276, 302, 306, 309, and 322) or metH only (residues 258, 264, 290, 294, and 295). The second is a distinct cluster of residues important for metE activation only (residues 285, 289, 313, and 314). We propose that a difference in the location of the MetR binding site for activation at these two promoters accounts for the differences in the residues of α required for MetR-dependent activation. We have designed an in vitro reconstitution-purification protocol that allows us to specifically orient wild-type or mutant α subunits to either the β-associated or the β′-associated position within RNAP (comprising α2, β, β′, and ς subunits). In vitro transcriptions using oriented α RNAP indicate that a single αCTD on either the β- or the β′-associated α subunit is sufficient for MetR activation of metE, while MetR interacts preferentially with the αCTD on the β-associated α subunit at metH. We propose that the different αCTD requirements at these two promoters are due to a combination of the difference in the location of the activation site and limits on the rotational flexibility of the αCTD.

scholarly journals A study of the mechanism of lncRNA-CR594175 in regulating proliferation and invasion of hepatocellular carcinoma cells in vivo and in vitro

2020 ◽  
Author(s):  
Quan Liu ◽  
Xuxu Yu ◽  
Minjie Yang ◽  
Xiangke Li ◽  
Xuejia Zhai ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
High Throughput Screening ◽  
Hepatoma Cell ◽  
Screening Method ◽  
Negative Regulation ◽  
Hepatoma Cell Line ◽  
Incidence And Mortality ◽  
Proliferation And Invasion

Abstract Abstract Background Hepatocellular carcinoma (HCC) is one of the cancers of highest incidence and mortality worldwide. The proliferation and invasion of tumor cells are the main reason for poor prognosis after HCC surgery. Long non-coding RNA (lncRNA) has been shown to play a key role in the progression of HCC. LncRNA-CR594175 is one of the highly expressed lncRNAs in HCC tumors and their metastatic tumors that we have obtained by the High-throughput screening method.Methods To elucidate the role of lncRNA-CR594175 in regulating the proliferation and invasion of human hepatoma cell line, HepG2, we operated through lncRNA-CR594175 silencing to inhibit the progression of HCC, either through in vitro or in vivo experiments.Results We found that lncRNA-CR594175 was lower in adjacent non-cancerous tissues than in primary HCC, and was lower in primary HCC than in its metastasis. Silencing of lncRNA-CR594175 inhibited the proliferation and invasion of HepG2 cells and growth of subcutaneous tumors. The results revealed that lncRNA-CR594175, as a RNA sponge, broke the negative regulation of hsa-miR-142-3p on Catenin, beta-1 (CTNNB1), and once lncRNA-CR594175 was silenced, the hsa-miR142-3p regained its negative regulation on CTNNB1 which can promote HCC progression by activating the wnt pathway. Conclusions Our present study demonstrated for the first time that lncRNA-CR594175 silencing suppressed proliferation and invasion of HCC cells in vivo and in vitro by restoring the negative regulation of hsa-miR-142-3p on CTNNB1, laying a solid theoretical base for using lncRNA-CR594175 as genetic target therapy for HCC and offering a reasonable explanation for inactivation of miRNA in different tumors or in the tumor at different stages.

scholarly journals Development of a Simple In Vitro Assay To Identify and Evaluate Nucleotide Analogs against SARS-CoV-2 RNA-Dependent RNA Polymerase

2020 ◽  
Vol 65 (1) ◽  
pp. e01508-20
Author(s):  
Gaofei Lu ◽  
Xi Zhang ◽  
Weinan Zheng ◽  
Jialei Sun ◽  
Lan Hua ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Antiviral Treatment ◽  
Screening Method ◽  
Chain Termination ◽  
Rna Dependent Rna Polymerase ◽  
Nucleotide Analogs ◽  
Vitro Assay ◽  
Assay Conditions ◽  
Incorporation Efficiency

ABSTRACTNucleotide analogs targeting viral RNA polymerase have been proved to be an effective strategy for antiviral treatment and are promising antiviral drugs to combat the current severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. In this study, we developed a robust in vitro nonradioactive primer extension assay to quantitatively evaluate the efficiency of incorporation of nucleotide analogs by SARS-CoV-2 RNA-dependent RNA polymerase (RdRp). Our results show that many nucleotide analogs can be incorporated into RNA by SARS-CoV-2 RdRp and that the incorporation of some of them leads to chain termination. The discrimination values of nucleotide analogs over those of natural nucleotides were measured to evaluate the incorporation efficiency of nucleotide analog by SARS-CoV-2 RdRp. In agreement with the data published in the literature, we found that the incorporation efficiency of remdesivir-TP is higher than that of ATP and incorporation of remdesivir-TP caused delayed chain termination, which can be overcome by higher concentrations of the next nucleotide to be incorporated. Our data also showed that the delayed chain termination pattern caused by remdesivir-TP incorporation is different for different template sequences. Multiple incorporations of remdesivir-TP caused chain termination under our assay conditions. Incorporation of sofosbuvir-TP is very low, suggesting that sofosbuvir may not be very effective in treating SARS-CoV-2 infection. As a comparison, 2′-C-methyl-GTP can be incorporated into RNA efficiently, and the derivative of 2′-C-methyl-GTP may have therapeutic application in treating SARS-CoV-2 infection. This report provides a simple screening method that should be useful for evaluating nucleotide-based drugs targeting SARS-CoV-2 RdRp and for studying the mechanism of action of selected nucleotide analogs.

scholarly journals High-throughput screening of biomolecules using cell-free gene expression systems

2018 ◽  
Vol 3 (1) ◽  
Author(s):  
Luis E Contreras-Llano ◽  
Cheemeng Tan
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Screening Method ◽  
Screening Methods ◽  
Free Gene ◽  
On Chip ◽  
Translation Systems ◽  
Selection Of

Abstract The incorporation of cell-free transcription and translation systems into high-throughput screening applications enables the in situ and on-demand expression of peptides and proteins. Coupled with modern microfluidic technology, the cell-free methods allow the screening, directed evolution and selection of desired biomolecules in minimal volumes within a short timescale. Cell-free high-throughput screening applications are classified broadly into in vitro display and on-chip technologies. In this review, we outline the development of cell-free high-throughput screening methods. We further discuss operating principles and representative applications of each screening method. The cell-free high-throughput screening methods may be advanced by the future development of new cell-free systems, miniaturization approaches, and automation technologies.

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