Investigation and Structural Analysis of Innate Immune Sensor PKR Activation by the Clostridia Bolteae Twister Ribozyme

Open Access
Anmangandla, Ananya
Area of Honors:
Bachelor of Science
Document Type:
Thesis Supervisors:
  • Philip Bevilacqua, Thesis Supervisor
  • Raymond Funk, Honors Advisor
  • RNA
  • Ribozyme
  • PKR
  • Protein Kinase R
  • Innate Immunity
  • Biochemistry
  • Chemistry
  • Functional RNAs
  • Twister Ribozyme
The innate immune system is the body’s first line of defense. This system uses a generalized mechanism to differentiate cellular RNA from pathogenic RNA like that of bacteria and viruses. The RNA-activated protein kinase, PKR, binds dsRNA and is a necessary sensor in the innate immune response. PKR is known to recognize long stretches of viral dsRNA and inhibit translation by autophosphorylation and subsequent phosphorylation of initiation factor eIF2α. Recently, PKR has been found to be activated by a wider range of pathogens, including certain bacteria. We studied the interaction between PKR and the self-cleaving, structurally compact C. bolteae twister ribozyme. This RNA is representative of small, structured RNAs that are typically found in bacteria. I found that as 3’ tail length of the RNA increases, PKR activation becomes more potent. Further, I find that in the presence of lowered Mg2+, similar to when bacterial RNA enters the human cell, activation of PKR by the short, compact ribozyme still occurs, albeit two-fold lower, supporting the hypothesis that PKR potentially is activated by this or similar RNAs in vivo. Following these findings, the interaction between PKR and the twister ribozyme was further characterized through PKR footprinting. While no obvious PKR footprint is observed, we show that following PKR addition, tertiary structure of the RNA remains intact, consistent with previous characterizations of PKR’s interactions with functional RNAs. This may indicate PKR is binding nonspecific helical regions of the RNA. My results represent a model system for similar RNAs in vivo and provide new insights into how PKR acts as an innate immune signaling protein for the presence of bacteria and suggest a reason for the apparent absence of protein-free riboswitches and ribozymes in the human genome.