scholarly journals Mutational Analysis of the Role of Nucleoside Triphosphatase P4 in the Assembly of the RNA Polymerase Complex of Bacteriophage φ6

1998 ◽  
Vol 72 (12) ◽  
pp. 10058-10065 ◽  
Author(s):  
Anja O. Paatero ◽  
Leonard Mindich ◽  
Dennis H. Bamford
Keyword(s):  
Rna Polymerase ◽  
Mutational Analysis ◽  
Rna Virus ◽  
Nucleoside Triphosphate ◽  
Particle Assembly ◽  
Polymerase Complex ◽  
Translocation Event ◽  
Shell Forming ◽  
Nucleoside Triphosphatase

ABSTRACT Bacteriophage φ6 is a complex enveloped double-stranded RNA virus with a segmented genome and replication strategy quite similar to that of the Reoviridae. An in vitro packaging and replication system using purified components is available. The positive-polarity genomic segments are translocated into a preformed polymerase complex (procapsid) particle. This particle is composed of four proteins: the shell-forming protein P1, the RNA polymerase P2, and two proteins active in packaging. Protein P7 is involved in stable packaging, and protein P4 is a homomultimeric potent nucleoside triphosphatase that provides the energy for the RNA translocation event. In this investigation, we used mutational analysis to study P4 multimerization and assembly. P4 is assembled onto a preformed particle containing proteins P2 and P7 in addition to P1. Only simultaneous production of P1 and P4 in the same cell leads to P4 assembly on P1 alone, whereas the P1 shell is incompetent for accepting P4 if produced separately. The C-terminal part of P4 is essential for particle assembly but not for multimerization or enzymatic activity. Altering the P4 nucleoside triphosphate binding site destroys the ability to form multimers.

scholarly journals Functional dissection of the catalytic mechanism of mammalian RNA polymerase II

2005 ◽  
Vol 83 (4) ◽  
pp. 497-504 ◽  
Author(s):  
Benoit Coulombe ◽  
Marie-France Langelier
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Catalytic Mechanism ◽  
Mutational Analysis ◽  
Structural Elements ◽  
Catalytic Mechanisms ◽  
Rnap Ii ◽  
Affinity Peptide

High resolution X-ray crystal structures of multisubunit RNA polymerases (RNAP) have contributed to our understanding of transcriptional mechanisms. They also provided a powerful guide for the design of experiments aimed at further characterizing the molecular stages of the transcription reaction. Our laboratory used tandem-affinity peptide purification in native conditions to isolate human RNAP II variants that had site-specific mutations in structural elements located strategically within the enzyme's catalytic center. Both in vitro and in vivo analyses of these mutants revealed novel features of the catalytic mechanisms involving this enzyme.Key words: RNA polymerase II, transcriptional mechanisms, mutational analysis, mRNA synthesis.

scholarly journals RNA-Dependent RNA Polymerase from Heterobasidion RNA Virus 6 Is an Active Replicase In Vitro

Viruses ◽  
2021 ◽  
Vol 13 (9) ◽  
pp. 1738
Author(s):  
Alesia A. Levanova ◽  
Eeva J. Vainio ◽  
Jarkko Hantula ◽  
Minna M. Poranen
Keyword(s):  
Rna Polymerase ◽  
Rna Synthesis ◽  
Rna Virus ◽  
Polymerization Reaction ◽  
Rna Dependent Rna Polymerase ◽  
Independent Manner ◽  
Nucleotidyl Transferase ◽  
Rna Polymerization ◽  
The Family

Heterobasidion RNA virus 6 (HetRV6) is a double-stranded (ds)RNA mycovirus and a member of the recently established genus Orthocurvulavirus within the family Orthocurvulaviridae. The purpose of the study was to determine the biochemical requirements for RNA synthesis catalyzed by HetRV6 RNA-dependent RNA polymerase (RdRp). HetRV6 RdRp was expressed in Escherichia coli and isolated to near homogeneity using liquid chromatography. The enzyme activities were studied in vitro using radiolabeled UTP. The HetRV6 RdRp was able to initiate RNA synthesis in a primer-independent manner using both virus-related and heterologous single-stranded (ss)RNA templates, with a polymerization rate of about 46 nt/min under optimal NTP concentration and temperature. NTPs with 2′-fluoro modifications were also accepted as substrates in the HetRV6 RdRp-catalyzed RNA polymerization reaction. HetRV6 RdRp transcribed viral RNA genome via semi-conservative mechanism. Furthermore, the enzyme demonstrated terminal nucleotidyl transferase (TNTase) activity. Presence of Mn2+ was required for the HetRV6 RdRp catalyzed enzymatic activities. In summary, our study shows that HetRV6 RdRp is an active replicase in vitro that can be potentially used in biotechnological applications, molecular biology, and biomedicine.

scholarly journals The human H5N1 influenza A virus polymerase complex is active in vitro over a broad range of temperatures, in contrast to the WSN complex, and this property can be attributed to the PB2 subunit

2008 ◽  
Vol 89 (12) ◽  
pp. 2923-2932 ◽  
Author(s):  
Birgit G. Bradel-Tretheway ◽  
Z. Kelley ◽  
Shikha Chakraborty-Sett ◽  
Toru Takimoto ◽  
Baek Kim ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Influenza A Virus ◽  
Influenza A ◽  
Elevated Temperatures ◽  
Expression System ◽  
Lung Epithelial Cells ◽  
Upper Respiratory Tract ◽  
Polymerase Complex ◽  
H5n1 Influenza

Influenza A virus (IAV) replicates in the upper respiratory tract of humans at 33 °C and in the intestinal tract of birds at close to 41 °C. The viral RNA polymerase complex comprises three subunits (PA, PB1 and PB2) and plays an important role in host adaptation. We therefore developed an in vitro system to examine the temperature sensitivity of IAV RNA polymerase complexes from different origins. Complexes were prepared from human lung epithelial cells (A549) using a novel adenoviral expression system. Affinity-purified complexes were generated that contained either all three subunits (PA/PB1/PB2) from the A/Viet/1203/04 H5N1 virus (H/H/H) or the A/WSN/33 H1N1 strain (W/W/W). We also prepared chimeric complexes in which the PB2 subunit was exchanged (H/H/W, W/W/H) or substituted with an avian PB2 from the A/chicken/Nanchang/3-120/01 H3N2 strain (W/W/N). All complexes were functional in transcription, cap-binding and endonucleolytic activity. Complexes containing the H5N1 or Nanchang PB2 protein retained transcriptional activity over a broad temperature range (30–42 °C). In contrast, complexes containing the WSN PB2 protein lost activity at elevated temperatures (39 °C or higher). The E627K mutation in the avian PB2 was not required for this effect. Finally, the avian PB2 subunit was shown to confer enhanced stability to the WSN 3P complex. These results show that PB2 plays an important role in regulating the temperature optimum for IAV RNA polymerase activity, possibly due to effects on the functional stability of the 3P complex.

scholarly journals Identifying SARS-CoV-2 Antiviral Compounds by Screening for Small Molecule Inhibitors of Nsp12/7/8 RNA-dependent RNA Polymerase

2021 ◽  
Author(s):  
Agustina P. Bertolin ◽  
Florian Weissmann ◽  
Jingkun Zeng ◽  
Viktor Posse ◽  
Jennifer C. Milligan ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Virus ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Etiologic Agent ◽  
Host Cells ◽  
Rdrp Activity ◽  
Chemical Library ◽  
Rna Dependent Rna Polymerase

SummaryThe coronavirus disease 2019 (COVID-19) global pandemic has turned into the largest public health and economic crisis in recent history impacting virtually all sectors of society. There is a need for effective therapeutics to battle the ongoing pandemic. Repurposing existing drugs with known pharmacological safety profiles is a fast and cost-effective approach to identify novel treatments. The COVID-19 etiologic agent is the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a single-stranded positive-sense RNA virus. Coronaviruses rely on the enzymatic activity of the replication-transcription complex (RTC) to multiply inside host cells. The RTC core catalytic component is the RNA-dependent RNA polymerase (RdRp) holoenzyme. The RdRp is one of the key druggable targets for CoVs due to its essential role in viral replication, high degree of sequence and structural conservation and the lack of homologs in human cells. Here, we have expressed, purified and biochemically characterised active SARS-CoV-2 RdRp complexes. We developed a novel fluorescence resonance energy transfer (FRET)-based strand displacement assay for monitoring SARS-CoV-2 RdRp activity suitable for a high-throughput format. As part of a larger research project to identify inhibitors for all the enzymatic activities encoded by SARS-CoV-2, we used this assay to screen a custom chemical library of over 5000 approved and investigational compounds for novel SARS-CoV-2 RdRp inhibitors. We identified 3 novel compounds (GSK-650394, C646 and BH3I-1) and confirmed suramin and suramin-like compounds as in vitro SARS-CoV-2 RdRp activity inhibitors. We also characterised the antiviral efficacy of these drugs in cell-based assays that we developed to monitor SARS-CoV-2 growth.

scholarly journals Mutational Analysis of an RNA Polymerase II Elongation Factor in Drosophila melanogaster

2005 ◽  
Vol 25 (17) ◽  
pp. 7803-7811 ◽  
Author(s):  
Mark A. Gerber ◽  
Ali Shilatifard ◽  
Joel C. Eissenberg
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Cultured Cells ◽  
Mutational Analysis ◽  
Elongation Factor ◽  
Regulatory Function ◽  
Polymerase Binding ◽  
Necessary And Sufficient ◽  
Elongation Activity

ABSTRACT The ELL family of proteins function in vitro as elongation factors for RNA polymerase II. Deletion studies have defined domains in mammalian ELL required for transcription elongation activity and RNA polymerase binding in vitro, for transformation of cultured cells when overexpressed, and for leukemogenesis and cell proliferation as part of a leukemic fusion protein. The goal of this study was to identify domains required for chromosome targeting and viability in the unique Drosophila ELL (dELL) protein. Here, we show that an N-terminal domain of dELL is necessary and sufficient for targeting to transcriptionally active puff sites in chromatin, supporting a role for this domain in recruiting dELL to elongating RNA polymerase II. We demonstrate that a central domain of dELL is required for rapid mobilization of ELL during the heat shock response, suggesting a regulatory function for this domain. Unexpectedly, transgenic dELL in which the N-terminal chromosome binding domain is deleted can complement the recessive lethality of mutations in ELL, suggesting that Drosophila ELL has an essential activity in development distinct from its role as an RNA polymerase II elongation factor.

Mutational analysis of the PA and PB2 subunits of the influenza RNA polymerase complex leads to new insights into function

2004 ◽  
Vol 1263 ◽  
pp. 25-28
Author(s):  
Ervin Fodor ◽  
Pierre Fechter ◽  
Mandy Crow ◽  
Tao Deng ◽  
Louise Mingay ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Mutational Analysis ◽  
Polymerase Complex

scholarly journals Concentration of 2’C-methyladenosine triphosphate byLeishmania guyanensisenables specific inhibition ofLeishmaniaRNA virus 1 via its RNA polymerase

2018 ◽  
Author(s):  
John I. Robinson ◽  
Stephen M. Beverley
Keyword(s):  
Rna Polymerase ◽  
Rna Virus ◽  
Protozoan Parasite ◽  
Cesium Chloride ◽  
Rdrp Activity ◽  
Cellular Mechanisms ◽  
The Impact ◽  
Leishmania Guyanensis ◽  
Good Agreement

AbstractLeishmaniais a widespread trypanosomatid protozoan parasite causing significant morbidity and mortality in humans. The endobiont dsRNA virusLeishmaniaRNA virus 1 (LRV1) chronically infects some strains, where it increases parasite numbers and virulence in murine leishmaniasis models, and correlates with increased treatment failure in human disease. Previously, we reported that 2’-C-methyladenosine (2CMA) potently inhibited LRV1 inLeishmania guyanensis(Lgy) andL. braziliensis, leading to viral eradication at concentrations above 10 µM. Here we probed the cellular mechanisms of 2CMA inhibition, involving metabolism, accumulation and inhibition of the viral RNA dependent RNA polymerase (RDRP). Activation to 2CMA triphosphate (2CMATP) was required, as 2CMA showed no inhibition of RDRP activity from virions purified on cesium chloride gradients. In contrast, 2CMA-TP showed IC50s ranging from 150 to 910 µM, depending on the CsCl density of the virion (empty, ssRNA- and dsRNA-containing).Lgyparasites incubatedin vitrowith 10 µM 2CMA accumulated 2CMA-TP to 410 µM, greater than the most sensitive RDRP IC50 measured. Quantitative modeling showed good agreement between the degree of LRV1 RDRP inhibition and LRV1 levels. These results establish that 2CMA activity is due to its conversion to 2CMA-TP, which accumulates to levels that inhibit RDRP and cause LRV1 loss. This attests to the impact of the Leishmania purine uptake and metabolism pathways, which allow even a weak RDRP inhibitor to effectively eradicate LRV1 at micromolar concentrations. Future RDRP inhibitors with increased potency may have potential therapeutic applications for ameliorating the increased Leishmania pathogenicity conferred by LRV1.

scholarly journals Mutational Analysis of the Chlamydia trachomatis rRNA P1 Promoter Defines Four Regions Important for Transcription In Vitro

1998 ◽  
Vol 180 (9) ◽  
pp. 2359-2366 ◽  
Author(s):  
Ming Tan ◽  
Tamas Gaal ◽  
Richard L. Gourse ◽  
Joanne N. Engel
Keyword(s):  
Escherichia Coli ◽  
Chlamydia Trachomatis ◽  
Rna Polymerase ◽  
Mutational Analysis ◽  
In Vitro Transcription ◽  
Rna Polymerases ◽  
Rich Region ◽  
E Coli ◽  
Transcription In Vitro

ABSTRACT We have characterized the Chlamydia trachomatisribosomal promoter, rRNA P1, by measuring the effect of substitutions and deletions on in vitro transcription with partially purifiedC. trachomatis RNA polymerase. Our analyses indicate that rRNA P1 contains potential −10 and −35 elements, analogous toEscherichia coli promoters recognized by E-ς70. We identified a novel AT-rich region immediately downstream of the −35 region. The effect of this region was specific for C. trachomatis RNA polymerase and strongly attenuated by single G or C substitutions. Upstream of the −35 region was an AT-rich sequence that enhanced transcription by C. trachomatis and E. coli RNA polymerases. We propose that this region functions as an UP element.

Demonstration that the TyrR Protein and RNA Polymerase Complex Formed at the Divergent P3 Promoter Inhibits Binding of RNA Polymerase to the Major Promoter, P1, of the aroP Gene ofEscherichia coli

1998 ◽  
Vol 180 (20) ◽  
pp. 5466-5472 ◽  
Author(s):  
Peixiang Wang ◽  
Ji Yang ◽  
Akira Ishihama ◽  
A. J. Pittard
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Regulatory Region ◽  
Ofescherichia Coli ◽  
Mrna Synthesis ◽  
Polymerase Complex ◽  
Defective Mutant ◽  
Dna Fragment ◽  
Region Ii

ABSTRACT In previous studies, we have identified three promoters (P1, P2, and P3) in the regulatory region of the Escherichia coli aroP gene (P. Wang, J. Yang, and A. J. Pittard, J. Bacteriol. 179:4206–4212, 1997). Both P1 and P2 can direct mRNA synthesis for aroP expression, whereas P3 is a divergent promoter which overlaps with P1. The repression of transcription from the major promoter, P1, has been postulated to involve the activation of the divergent promoter, P3, by the TyrR protein (P. Wang, J. Yang, B. Lawley, and A. J. Pittard, J. Bacteriol. 179:4213–4218, 1997). In the present study, we confirmed the proposed mechanism of P3-mediated repression of P1 transcription by studying the binding of RNA polymerase to the promoters P1 and P3 in vitro in the presence and absence of TyrR protein and its cofactors. Our results show that (i) only one RNA polymerase molecule can bind to the DNA fragment carrying the aroP regulatory region, (ii) RNA polymerase has a higher affinity for P1 than for either P2 or P3 and binds to P1 in the absence of TyrR protein, (iii) in the presence of TyrR protein and its cofactor, phenylalanine or tyrosine, RNA polymerase preferentially binds to P3, and (iv) RNA polymerase does not respond to the activation-defective mutant TyrR protein TyrR-RQ10 and remains bound to P1 in the presence of TyrR-RQ10 and either of the cofactors.

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