Regulation of interferon inducible Ser/Thr RNA-dependent protein kinase (PKR) by short imperfectly base-paired viral dsRNAs
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Date
2014-01, 2013-03
Authors
Dzananovic, Edis
Journal Title
Journal ISSN
Volume Title
Publisher
Journal of Structural Biology
RNA
RNA
Abstract
In response to viral infection cells produce the interferon inducible Ser/Thr RNAdependent
protein kinase (PKR) that binds viral dsRNAs. After initial binding, PKR selfassociates
and then becomes autophosphorylated. PKR then phosphorylates its substrate,
eukaryotic initiation factor 2α, which slows viral protein translation, thus helping the host
cell response. PKR consists of tandem double stranded RNA binding motifs (dsRBMs)
connected via a flexible linker to a kinase domain. A number of studies involving
individual dsRBMs from proteins other than PKR have highlighted the key features
required for interaction with perfectly duplexed RNA. However, viral dsRNA molecules
are highly structured and often contain deviations from perfect RNA helices. HIV-1 TAR
and adenovirus VAI RNAs are well-characterized PKR binding partners. HIV-1 is an
activator of PKR that adopts a mostly double-stranded structure with distortions
including a trinucleotide bulge and hexaloop. Adenovirus VAI RNA has double-stranded
secondary structural elements including a dsRNA-binding (apical stem), an inhibitory
stem-loop (central stem) that inhibits PKR from performing its enzymatic reaction, and
the terminal stem. A truncated version of VAI lacking the terminal stem called VAIΔTS
(often used as wild type RNA in this study), binds to PKR and prevent its selfassociation.
The interaction and binding affinities of PKR with all RNAs was determined
using electrophoretic mobility shift assays. To investigate the role of the central stemloop
in the mechanism of inhibition of PKR by the VAIΔTS RNA, truncated versions of
VAIΔTS with mutations in the central stem were transcribed. In vitro studies that include
well-established enzymatic assays test activation and inhibition of PKR in presence of the
ii
mutant versions of VAIΔTS. The solution conformations of the dsRBMs of PKR in
complex with TAR and VAIΔTS determined using small-angle X-ray scattering studies
show dsRBMs of PKR interact with both stem and loop regions of the RNAs. SAXS
modeling of VAIΔTS, mutant RNAs together with activation assays show that loop and
bulge regions are crucial for the tertiary structural integrity and function of central stem.
Taken together this data provides framework for the recognition of imperfectly basepaired
viral dsRNA by PKR and PKR’s regulation through RNA tertiary structure.
Description
Keywords
PKR: RNA-activated Protein Kinase, VAI: adenovirus virus-associated RNA, Innate immunity, dsRBMs: double-stranded RNA binding motifs, EMSA: electrophoretic mobility shift assays, DLS: dynamic light scattering, SAXS: small angle X-ray scattering
Citation
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