Regulation of interferon inducible Ser/Thr RNA-dependent protein kinase (PKR) by short imperfectly base-paired viral dsRNAs
| dc.contributor.author | Dzananovic, Edis | |
| dc.contributor.examiningcommittee | Stetefeld, Jorg (Chemistry) Sorensen, John (Chemistry) Brassinga, Ann Karen (Microbiology) MacMillan, Andrew (Biochemistry) | en_US |
| dc.contributor.supervisor | McKenna, Sean (Chemistry) | en_US |
| dc.date.accessioned | 2016-07-27T19:04:28Z | |
| dc.date.available | 2016-07-27T19:04:28Z | |
| dc.date.issued | 2014-01 | en_US |
| dc.date.issued | 2013-03 | en_US |
| dc.degree.discipline | Chemistry | en_US |
| dc.degree.level | Doctor of Philosophy (Ph.D.) | en_US |
| dc.description.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. | en_US |
| dc.description.note | October 2016 | en_US |
| dc.identifier.citation | ACS | en_US |
| dc.identifier.uri | http://hdl.handle.net/1993/31534 | |
| dc.language.iso | eng | en_US |
| dc.publisher | Journal of Structural Biology | en_US |
| dc.publisher | RNA | en_US |
| dc.rights | open access | en_US |
| dc.subject | PKR: RNA-activated Protein Kinase | en_US |
| dc.subject | VAI: adenovirus virus-associated RNA | en_US |
| dc.subject | Innate immunity | en_US |
| dc.subject | dsRBMs: double-stranded RNA binding motifs | en_US |
| dc.subject | EMSA: electrophoretic mobility shift assays | en_US |
| dc.subject | DLS: dynamic light scattering | en_US |
| dc.subject | SAXS: small angle X-ray scattering | en_US |
| dc.title | Regulation of interferon inducible Ser/Thr RNA-dependent protein kinase (PKR) by short imperfectly base-paired viral dsRNAs | en_US |
| dc.type | doctoral thesis | en_US |