Characterizing retron Efe1 reverse transcriptase interactions with ncRNA

dc.contributor.advisorFinkelstein, Ilya J.
dc.creatorArguello, Carlos
dc.date.accessioned2024-04-29T13:01:25Z
dc.date.available2024-04-29T13:01:25Z
dc.date.issued2024-04
dc.description.abstractRetrons are diverse bacterial anti-phage defense systems. A retron operon consists of a reverse transcriptase, an accessory protein, and a structured non-coding RNA that serves as the primer and template for reverse transcription. Retrons are currently being developed into new gene editing tools in bacteria, plants, and mammalian cells. A new retron system discovered in the Finkelstein lab, Efe1, has shown higher gene editing rates in mammalian cells than the current standard in retron gene editing, Eco1. Discovering what makes Efe1 better than Eco1 can elucidate the molecular mechanisms behind retron functions. Here, I investigate Efe1 reverse transcriptase and use cryo-electron microscopy to reconstruct a 3.9 Å density map of its RT-msDNA complex. Efe1 complex is highly similar to Eco1 complex, except that it is a monomer and its msDNA has a more rigid DNA stem loop than Eco1. Efe1 reverse transcriptase solubility decreases drastically in the absence of its cognate ncRNA. Efe1 reverse transcriptase can also be solubilized by Eco1 ncRNA and produce Eco1 msDNA. Mutating catalytic residues in Efe1 reverse transcriptase ablates msDNA production and reduces solubility. These findings offer insight into retron reverse transcriptase interactions with ncRNA that dictate proper protein folding and provide some guidance for future attempts at purifying retron reverse transcriptase alone.
dc.description.departmentMolecular Biosciences
dc.identifier.urihttps://hdl.handle.net/2152/124938
dc.identifier.urihttps://doi.org/10.26153/tsw/51540
dc.language.isoen
dc.relation.ispartofHonors Thesesen
dc.rights.restrictionOpen
dc.subjectRetron
dc.subjectEfe1
dc.subjectbacteria
dc.subjectanti-phage defense
dc.subjectreverse transcription
dc.subjectreverse transcriptase
dc.subjectcryo-EM
dc.subjectstructure-function
dc.subjectexpression
dc.titleCharacterizing retron Efe1 reverse transcriptase interactions with ncRNA
dc.typeThesis

Access full-text files

Original bundle

Now showing 1 - 1 of 1
Loading...
Thumbnail Image
Name:
Arguello_Carlos_Honors_Thesis.pdf
Size:
1.46 MB
Format:
Adobe Portable Document Format

License bundle

Now showing 1 - 1 of 1
No Thumbnail Available
Name:
license.txt
Size:
1.64 KB
Format:
Item-specific license agreed upon to submission
Description:

Collections