title: Mechanistic Analysis of Activation of the Innate Immune Sensor PKR by Bacterial RNA.
abstract: The protein kinase PKR (protein kinase R) is a sensor in innate immunity. PKR autophosphorylates in the presence of double-stranded RNA enabling it to phosphorylate its substrate, eIF2α (eukaryotic initiation factor 2α), halting cellular translation. Classical activators of PKR are long viral double-stranded RNAs, but recently, PKR has been found to be activated by bacterial RNA. However, the features of bacterial RNA that activate PKR are unknown. We studied the Bacillus subtilis trp 5'-UTR (untranslated region), which is an indirect riboswitch with secondary and tertiary RNA structures that regulate gene function. Additionally, the trp 5'-UTR binds a protein, TRAP (tryptophan RNA-binding attenuation protein), which recognizes l-tryptophan. We present the first evidence that multiple structural features in this RNA, which are typical of bacterial RNAs, activate PKR in TRAP-free and TRAP/l-Trp-bound forms. Segments from the 5'-UTR, including the terminator 5'-stem-loop and Shine-Dalgarno blocking hairpins, demonstrated 5'-triphosphate and flanking RNA tail dependence on PKR activation. Disruption of long-distance tertiary interactions in the 5'-UTR led to partial loss in activation, consistent with highly base-paired regions in bacterial RNA activating PKR. One physiological change a bacterial RNA would face in a human cell is a decrease in the concentration of free magnesium. Upon lowering the magnesium concentration to human physiological conditions of 0.5mM, the trp 5'-UTR continued to activate PKR potently. Moreover, total RNA from Escherichia coli, depleted of rRNA, also activated PKR under these ionic conditions. This study demonstrates that PKR can signal the presence of bacterial RNAs under physiological ionic conditions and offers a potential explanation for the apparent absence of riboswitches in the human genome.
authors.affiliation: Department of Chemistry, Center for RNA Molecular Biology, Pennsylvania State University, University Park, PA 16802, USA.
authors.forename: Chelsea M
authors.initials: CM
authors.lastname: Hull
authors.affiliation: Department of Chemistry, Center for RNA Molecular Biology, Pennsylvania State University, University Park, PA 16802, USA. Electronic address: pcb5@psu.edu.
authors.forename: Philip C
authors.initials: PC
authors.lastname: Bevilacqua
mesh_terms: D020121:5' Untranslated Regions / Q000235:genetics*; D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D020029:Base Pairing; D001483:Base Sequence; D004789:Enzyme Activation; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D008274:Magnesium / Q000494:pharmacology; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010766:Phosphorylation; D011485:Protein Binding; D012329:RNA, Bacterial / Q000235:genetics*; D012330:RNA, Double-Stranded / Q000235:genetics*; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D014364:Tryptophan / Q000378:metabolism; D019892:eIF-2 Kinase / Q000235:genetics / Q000378:metabolism*
chemical_list: D020121:5' Untranslated Regions; D001426:Bacterial Proteins; C079092:MtrB protein, Bacteria; D012329:RNA, Bacterial; D012330:RNA, Double-Stranded; D016601:RNA-Binding Proteins; D014157:Transcription Factors; D014364:Tryptophan; D019892:eIF-2 Kinase; D008274:Magnesium
keywords: PKR; RNA folding; bacterial RNA; innate immunity; riboswitch