RNA-Binding to Archaeal RNA Polymerase Subunits F/E: A DEER and FRET Study

AbstractMammalian reovirus (MRV) is the prototypical member of genus Orthoreovirus of family Reoviridae. However, lacking high-resolution structures of its RNA polymerase cofactor μ2 and infectious particle, limits understanding of molecular interactions among proteins and RNA, and their contributions to virion assembly and RNA transcription. Here, we report the 3.3 Å-resolution asymmetric reconstruction of transcribing MRV and in situ atomic models of its capsid proteins, the asymmetrically attached RNA-dependent RNA polymerase (RdRp) λ3, and RdRp-bound nucleoside triphosphatase μ2 with a unique RNA-binding domain. We reveal molecular interactions among virion proteins and genomic and messenger RNA. Polymerase complexes in three Spinoreovirinae subfamily members are organized with different pseudo-D3d symmetries to engage their highly diversified genomes. The above interactions and those between symmetry-mismatched receptor-binding σ1 trimers and RNA-capping λ2 pentamers balance competing needs of capsid assembly, external protein removal, and allosteric triggering of endogenous RNA transcription, before, during and after infection, respectively.

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Eukaryotic transcription termination factor La mediates transcript release and facilitates reinitiation by RNA polymerase III

Molecular and Cellular Biology ◽

10.1128/mcb.14.3.2147-2158.1994 ◽

1994 ◽

Vol 14 (3) ◽

pp. 2147-2158

Author(s):

R J Maraia ◽

D J Kenan ◽

J D Keene

Keyword(s):

Rna Polymerase ◽

Rna Binding ◽

Transcription Termination ◽

Rna Polymerase Iii ◽

Class Iii ◽

Ample Evidence ◽

Polymerase Iii ◽

Transcription Complexes ◽

Pol Iii

Ample evidence indicates that Alu family interspersed elements retrotranspose via primary transcripts synthesized by RNA polymerase III (pol III) and that this transposition sometimes results in genetic disorders in humans. However, Alu primary transcripts can be processed posttranscriptionally, diverting them away from the transposition pathway. The pol III termination signal of a well-characterized murine B1 (Alu-equivalent) element inhibits RNA 3' processing, thereby stabilizing the putative transposition intermediary. We used an immobilized template-based assay to examine transcription termination by VA1, 7SL, and Alu class III templates and the role of transcript release in the pol III terminator-dependent inhibition of processing of B1-Alu transcripts. We found that the RNA-binding protein La confers this terminator-dependent 3' processing inhibition on transcripts released from the B1-Alu template. Using pure recombinant La protein and affinity-purified transcription complexes, we also demonstrate that La facilitates multiple rounds of transcription reinitiation by pol III. These results illustrate an important role for La in RNA production by demonstrating its ability to clear the termination sites of class III templates, thereby promoting efficient use of transcription complexes by pol III. The role of La as a potential regulatory factor in transcript maturation and how this might apply to Alu interspersed elements is discussed.

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Conformational changes in human Norovirus polymerase

Acta Crystallographica Section A Foundations and Advances ◽

10.1107/s2053273314084046 ◽

2014 ◽

Vol 70 (a1) ◽

pp. C1595-C1595

Author(s):

Kenneth Ng ◽

Dmitry Zamyatkin ◽

Hayeong Rho ◽

Elesha Hoffarth ◽

Gabriela Jurca ◽

...

Keyword(s):

Rna Polymerase ◽

Conformational Changes ◽

Rna Binding ◽

Divalent Metal ◽

Rna Dependent Rna Polymerase ◽

Human Norovirus ◽

Conformational States ◽

Crystal Forms ◽

Divalent Metal Cations ◽

Positive Strand Rna

Human Noroviruses (NV) belong in the Caliciviridae family and are a major cause of gastroenteritis outbreaks throughout the world. Crystal structures of the RNA-dependent RNA polymerase from the human Norovirus have been determined in over ten different crystal forms in the presence and absence of divalent metal cations, nucleoside triphosphates, inhibitors and primer-template duplex RNA. These structures show how the polymerase enzyme can adopt a range of conformations in which the thumb, fingers and palm domains change orientations depending on the step of the enzymatic cycle trapped in different crystal forms. We discuss how the evidence from crystallographic and biochemical experiments combine to better understand how viral RNA polymerase enzymes from human Norovirus and related positive-strand RNA viruses can adopt a range of conformational states to facilitate RNA binding, NTP binding, catalysis, RNA translocation and pyrophosphate release. The detailed structural and mechanistic understanding of these conformational changes is important for providing a sound basis for understanding viral replication in general, as well as for the design of novel inhibitors capable of trapping the enzyme in specific conformational states.

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Involvement of Hsp90 in Assembly and Nuclear Import of Influenza Virus RNA Polymerase Subunits

Journal of Virology ◽

10.1128/jvi.01917-06 ◽

2006 ◽

Vol 81 (3) ◽

pp. 1339-1349 ◽

Cited By ~ 168

Author(s):

Tadasuke Naito ◽

Fumitaka Momose ◽

Atsushi Kawaguchi ◽

Kyosuke Nagata

Keyword(s):

Influenza Virus ◽

Rna Polymerase ◽

Nuclear Transport ◽

Intracellular Localization ◽

Viral Rna ◽

Polymerase Complex ◽

Virus Rna ◽

Binary Complexes ◽

Infected Cells ◽

Rna Polymerase Subunits

ABSTRACT Transcription and replication of the influenza virus RNA genome occur in the nuclei of infected cells through the viral RNA-dependent RNA polymerase consisting of PB1, PB2, and PA. We previously identified a host factor designated RAF-1 (RNA polymerase activating factor 1) that stimulates viral RNA synthesis. RAF-1 is found to be identical to Hsp90. Here, we examined the intracellular localization of Hsp90 and viral RNA polymerase subunits and their molecular interaction. Hsp90 was found to interact with PB2 and PB1, and it was relocalized to the nucleus upon viral infection. We found that the nuclear transport of Hsp90 occurs in cells expressing PB2 alone. The nuclear transport of Hsp90 was in parallel with that of the viral RNA polymerase binary complexes, either PB1 and PB2 or PB1 and PA, as well as with that of PB2 alone. Hsp90 also interacted with the binary RNA polymerase complex PB1-PB2, and it was dissociated from the PB1-PB2 complex upon its association with PA. Furthermore, Hsp90 could form a stable PB1-PB2-Hsp90 complex prior to the formation of a ternary polymerase complex by the assembly of PA in the infected cells. These results suggest that Hsp90 is involved in the assembly and nuclear transport of viral RNA polymerase subunits, possibly as a molecular chaperone for the polymerase subunits prior to the formation of a mature ternary polymerase complex.

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Two mutations in the C-terminal domain of influenza virus RNA polymerase PB2 enhance transcription by enhancing cap-1 RNA binding activity

Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms ◽

10.1016/j.bbagrm.2011.11.006 ◽

2012 ◽

Vol 1819 (1) ◽

pp. 78-83 ◽

Cited By ~ 7

Author(s):

Shijian Zhang ◽

Qiang Wang ◽

Jinlan Wang ◽

Kiyohisa Mizumoto ◽

Tetsuya Toyoda

Keyword(s):

Influenza Virus ◽

Rna Polymerase ◽

Rna Binding ◽

Binding Activity ◽

Terminal Domain ◽

Virus Rna

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The RNA polymerase subunits E/F from the Antarctic archaeon Methanococcoides burtonii bind to specific species of mRNA

Environmental Microbiology ◽

10.1111/j.1462-2920.2010.02385.x ◽

2010 ◽

Vol 13 (8) ◽

pp. 2039-2055 ◽

Cited By ~ 4

Author(s):

Davide De Francisci ◽

Stefano Campanaro ◽

Geoff Kornfeld ◽

Khawar S. Siddiqui ◽

Timothy J. Williams ◽

...

Keyword(s):

Rna Polymerase ◽

Specific Species ◽

The Antarctic ◽

Rna Polymerase Subunits

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Sequences at the 3′ Untranslated Region of Bamboo Mosaic Potexvirus RNA Interact with the Viral RNA-Dependent RNA Polymerase

Journal of Virology ◽

10.1128/jvi.75.6.2818-2824.2001 ◽

2001 ◽

Vol 75 (6) ◽

pp. 2818-2824 ◽

Cited By ~ 40

Author(s):

Cheng-Yen Huang ◽

Yih-Leh Huang ◽

Menghsiao Meng ◽

Yau-Heiu Hsu ◽

Ching-Hsiu Tsai

Keyword(s):

Rna Polymerase ◽

Untranslated Region ◽

Rna Binding ◽

Bovine Liver ◽

Viral Rna ◽

Mobility Shift ◽

Rna Dependent Rna Polymerase ◽

Stem Loop ◽

Competition Analysis ◽

Reading Frame

ABSTRACT The 3′ untranslated region (UTR) of bamboo mosaic potexvirus (BaMV) genomic RNA was found to fold into a series of stem-loop structures including a pseudoknot structure. These structures were demonstrated to be important for viral RNA replication and were believed to be recognized by the replicase (C.-P. Cheng and C.-H. Tsai, J. Mol. Biol. 288:555–565, 1999). Electrophoretic mobility shift and competition assays have now been used to demonstrate that theEscherichia coli-expressed RNA-dependent RNA polymerase domain (Δ893) derived from BaMV open reading frame 1 could specifically bind to the 3′ UTR of BaMV RNA. No competition was observed when bovine liver tRNAs or poly(I)(C) double-stranded homopolymers were used as competitors, and the cucumber mosaic virus 3′ UTR was a less efficient competitor. Competition analysis with different regions of the BaMV 3′ UTR showed that Δ893 binds to at least two independent RNA binding sites, stem-loop D and the poly(A) tail. Footprinting analysis revealed that Δ893 could protect the sequences at loop D containing the potexviral conserved hexamer motif and part of the stem of domain D from chemical cleavage.

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