scholarly journals Conformational changes in Lassa virus L protein associated with promoter binding and RNA synthesis activity

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Tomas Kouba ◽  
Dominik Vogel ◽  
Sigurdur R. Thorkelsson ◽  
Emmanuelle R. J. Quemin ◽  
Harry M. Williams ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Rna Synthesis ◽  
Hemorrhagic Fever ◽  
Polymerase Activity ◽  
Lassa Virus ◽  
L Protein ◽  
Active Site Cavity ◽  
Binding Pockets ◽  
Rna Polymerase Activity ◽  
Synthesis Activity

AbstractLassa virus is endemic in West Africa and can cause severe hemorrhagic fever. The viral L protein transcribes and replicates the RNA genome via its RNA-dependent RNA polymerase activity. Here, we present nine cryo-EM structures of the L protein in the apo-, promoter-bound pre-initiation and active RNA synthesis states. We characterize distinct binding pockets for the conserved 3’ and 5’ promoter RNAs and show how full-promoter binding induces a distinct pre-initiation conformation. In the apo- and early elongation states, the endonuclease is inhibited by two distinct L protein peptides, whereas in the pre-initiation state it is uninhibited. In the early elongation state, a template-product duplex is bound in the active site cavity together with an incoming non-hydrolysable nucleotide and the full C-terminal region of the L protein, including the putative cap-binding domain, is well-ordered. These data advance our mechanistic understanding of how this flexible and multifunctional molecular machine is activated.

scholarly journals Conformational changes in Lassa virus L protein associated with promoter binding and RNA synthesis activity

2021 ◽  
Author(s):  
Tomas Kouba ◽  
Dominik Vogel ◽  
Sigurdur R. Thorkelsson ◽  
Emmanuelle R. J. Quemin ◽  
Harry M. Williams ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Active Site ◽  
Rna Synthesis ◽  
Antiviral Drug ◽  
Structural Data ◽  
Lassa Virus ◽  
Genome Replication ◽  
L Protein ◽  
Active Site Cavity

Lassa virus, which causes annual outbreaks in West Africa with increasing case numbers in recent years, is recognized by the WHO R&D blueprint as a significant threat for public health with high epidemic potential and no effective countermeasures. The viral large (L) protein, which contains the RNA-dependent RNA polymerase, is a key player for transcription of viral mRNA and genome replication. Here we present nine cryo-EM structures of Lassa virus L protein in the apo-, promoter-bound pre-initiation and active RNA synthesis states. We characterize distinct binding pockets for the conserved genomic 3' and 5' promoter RNAs and show how full-promoter binding induces a distinct pre-initiation conformation. In the apo- and elongation states, the endonuclease is inhibited by the binding of two distinct L protein peptides in the active site, respectively, whereas in the pre-initiation state, the endonuclease is uninhibited. In the stalled, early elongation state, a template-product duplex is bound in the active site cavity together with an incoming non-hydrolysable nucleotide. In this configuration, the full C-terminal region of the L protein, including the putative cap-binding domain, is highly ordered. The structural data are complemented by in vitro and cell-based studies testing a broad range of L protein mutants to probe functional relevance. These data advance our mechanistic understanding of how this flexible and multifunctional molecular machine is activated and will underpin antiviral drug development targeting the arenavirus L protein.

scholarly journals RNA Polymerase Activity Assay on Biochips: Correlation between Template DNA Density and RNA Synthesis

2010 ◽  
Vol 31 (7) ◽  
pp. 2107-2109 ◽  
Author(s):  
Bok-Hui Lee ◽  
Hyun-Jung Seo ◽  
So-Hyun Kim ◽  
Woong Jung ◽  
Dong-Woon Kim ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Synthesis ◽  
Polymerase Activity ◽  
Activity Assay ◽  
Rna Polymerase Activity ◽  
Template Dna ◽  
Dna Density

In vitro transcription of RNA in nuclei, nucleoli and chromatin from physarum polycephalum

1977 ◽  
Vol 26 (1) ◽  
pp. 267-279
Author(s):  
K.E. Davies ◽  
I.O. Walker
Keyword(s):  
Rna Polymerase ◽  
Physarum Polycephalum ◽  
Rna Synthesis ◽  
In Vitro Transcription ◽  
Polymerase Activity ◽  
Controlled Conditions ◽  
Rna Polymerase Activity ◽  
Alpha Amanitin

Methods for isolating nuclei, nucleoli and chromatin from Physarum polycephalum which retain high levels of endogenous RNA polymerase activity are described. Under carefully controlled conditions with respect to mono- and divalent cation concentrations RNA synthesis in nuclei displayed linear kinetics for at least 30 min and the RNA products had a similar size distribution to nuclear RNA synthesis observed in vivo. Chromatin showed 60% of the nuclear transcriptional activity but no conditions were found where faithful transcription of the template occurred. Isolated nucleoli were 5-fold more active than nuclei and the endogenous RNA polymerase activity was insensitive to alpha-amanitin. Under carefully controlled conditions, the nucleoli appeared to support the accurate transcription, re-initiation and processing of rRNA chains in vitro.

Changes in RNA-Synthesizing Activity and Template Activity in Nuclei From Cotyledons of Developing Pea Seeds

1974 ◽  
Vol 1 (3) ◽  
pp. 331 ◽  
Author(s):  
A Millerd ◽  
D Spencer
Keyword(s):  
Rna Synthesis ◽  
Dry Weight ◽  
Polymerase Activity ◽  
Template Activity ◽  
Rna Polymerase Activity ◽  
Pea Seeds ◽  
Cotyledon Cells ◽  
Weight Number ◽  
Number Of Cells ◽  
Template Activity Of Chromatin

Using Pisum sativum grown under controlled conditions, the pattern of cotyledon development has been defined in terms of fresh and dry weight, number of cells, and content of chlorophyll, starch, DNA, RNA and protein. The onset of storage-protein synthesis has been determined using antibodies to purified vicilin and legumin. During growth by cell expansion, DNA per nucleus of the parenchymatous cotyledon cells increases to an average of 50 C. Measurements of the endogenous RNA polymerase activity of nuclei and the template activity of chromatin isolated at various stages during seed development, suggest that the DNA beyond 2 C level does not contribute in a major way to RNA synthesis, although its template activity per unit of DNA is not diminished.

scholarly journals Mechanism of inhibition of Escherichia coli RNA polymerase by captan

1982 ◽  
Vol 201 (1) ◽  
pp. 145-151 ◽  
Author(s):  
J W Dillwith ◽  
R A Lewis
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Rna Synthesis ◽  
Polymerase Activity ◽  
Mechanism Of Inhibition ◽  
Dna Binding Site ◽  
Rna Polymerase Activity ◽  
Dna Template ◽  
Template Dna

Captan (N-trichloromethylthiocyclohex-4-ene-1,2-dicarboximide) was shown to inhibit RNA synthesis in vitro catalysed by Escherichia coli RNA polymerase. Incorporation of [gamma-32P]ATP and [gamma-32P]GTP was inhibited by captan to the same extent as overall RNA synthesis. The ratio of [3H]UTP incorporation to that of [gamma-32P]ATP or of [gamma-32P]GTP in control and captan-treated samples indicated that initiation was inhibited, but the length of RNA chains being synthesized was not altered by captan treatment. Limited-substrate assays in which re-initiation of RNA chains did not occur also showed that captan had no effect on the elongation reaction. Studies which measured the interaction of RNA polymerase with template DNA revealed that the binding of enzyme to DNA was inhibited by captan. Glycerol-gradient sedimentation of the captan-treated RNA polymerase indicated that the inhibition of the enzyme was irreversible and did not result in dissociation of its subunits. These data are consistent with a mechanism in which RNA polymerase activity was irreversibly altered by captan, resulting in an inability of the enzyme to bind to the template. This interaction was probably at the DNA-binding site on the polymerase and did not involve reaction of captan with the DNA template.

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