Description
- Abstract:
- Abstract of Biophysical Analysis of the Primary Nucleic Acid Binding Domains of HsLINE-1 ORF1p, by Raphael Britt, Ph.D., Brown University, May 2026. Long Interspersed Nuclear Element-1s (LINE-1s or L1s) are the only fully autonomous retrotransposons still active in the human genome and require ORF1p, a trimeric nucleic acid binding protein, for productive mobilization. Although ORF1p is essential for L1 retrotransposition, the residue-level basis of its nucleic acid binding activity remains incompletely understood. In this study, targeted charge-neutralizing mutations across the HsORF1p RRM-CTD binding surface were used to test how basic residues contribute to nucleic acid binding, thermal stability, retrotransposition efficiency, and cellular foci formation. Spectral shift and DSF assays showed that most mutations reduced nucleic acid binding affinity, supporting a broad role for the positively charged RRM-CTD surface in productive ssNA engagement. Retrotransposition efficiency largely tracked with binding affinity, while gross HsORF1p foci formation was less sensitive to mutation, suggesting that visible foci are not sufficient to predict retrotransposition competence. DSF measurements further showed that nucleic acid identity, especially poly(A) RNA, strongly influenced HsORF1p stabilization across mutant backgrounds. Together, these results support a model in which basic residues across the HsORF1p RRM-CTD contribute overlapping functions in nucleic acid engagement and L1 mobilization, while ligand-dependent stabilization not only requires a negatively charged sugar-phosphate backbone but is also significantly influenced by nitrogenous-base composition and positioning.
- Notes:
- Thesis (Ph. D.)--Brown University, 2026
Citation
Britt, Raphael Isaiah,
"Biophysical Analysis of the Primary Nucleic Acid Binding Domains of HsLINE-1 ORF1p"
(2026).
Molecular Biology, Cell Biology, and Biochemistry Theses and Dissertations.
Brown Digital Repository. Brown University Library.
https://repository.library.brown.edu/studio/item/bdr:pnbhv68j/
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Molecular Biology, Cell Biology, and Biochemistry Theses and Dissertations
Theses and Dissertations for the Molecular Biology, Cell Biology, and Biochemistry department....