Super-Resolution Force Spectroscopy for Revealing Sub-nucleotide Motion in Ribosomal Frameshifting and DNA Methylation

Programmed ribosomal frameshifting plays an important role for many viruses in their protein synthesis, with well-known cases including HIV and SARS-CoV-2. Aiming for an approach to defend against such viruses, techniques including X-ray crystallography, cryo-electron microscopy (Cryo-EM) and fluorescent resonance energy transfer (FRET) have been utilized to study the translocation mechanism. However, it remains challenging to directly resolve the different reading frames. This dissertation focuses on applying super-resolution force spectroscopy (SURFS) as a unique tool to resolve frameshifting products and intermediate states, with unprecedented resolution. Two different forms of forces have been studied, centrifugal force and acoustic radiation force. Using centrifugal force with 2 pN resolution that is sufficient to resolve a single nucleotide difference, we were able to reveal the efficiency of SufB2-suppressed frameshifting. Combined with collaborators’ kinetic measurements, our results identified twice exploration of +1 frameshifting in an elongation cycle, one during translocation from the aminoacyl-tRNA binding (A) site to the peptidyl-tRNA binding (P) site and the second during occupancy in the P site. Using acoustic radiation force, the force resolution was improved to 0.5 pN, sufficient to resolve a single hydrogen bond difference. Taking the advantage of the precision, we observed a new intermediate state in frameshifting induced by the GA7G slippery motif beside its original “-1” and “-2” frameshifting complexes in the presence of fusidic acid. We termed this new state as “-1*”, whose dissociation force was shifted by approximately half a nucleotide compared to the normal “-1” reading frame at the 5’-end of the mRNA. With this unprecedented force resolution, we were also able distinguished DNA duplexes with 1 and 2 methylated cytosine compared with the nonmethylated duplexes. Moreover, interactions between d-/l-THP and DNA duplexes were studied. We found that single-methylation only partly limits the interaction between l-THP and DNA, when leaving d-THP – DNA interaction changed. We have utilized SURFS as a uniquely capable tool in those biophysical systems with small stability differences, where no other force techniques have sufficient resolution to deal with. Broader applications of SURFS are expected in both fundamental and applied biological research.

Name thesis:degree_name

Doctor of Philosophy

Level thesis:degree_level

Doctoral

Discipline thesis:degree_discipline

Chemistry

Grantor

University of Houston

Year dc:date.issued

2021

Author dc:creator

• Mao, Yujia

Advisor dc:contributor.advisor

• Xu, Shoujun

Committee members dc:contributor.committeemember

• Baldelli, Steven
• Chen, Tai-Yen
• Harth, Eva M.
• Zhang, Xiaoliu Shaun

• Antibiotics
• DNA rulers
• Force spectroscopy
• Ribosomal frameshift
• DNA methylation

Statement dc:rights

• The author of this work is the copyright owner. UH Libraries and the Texas Digital Library have their permission to store and provide access to this work. UH Libraries has secured permission to reproduce any and all previously published materials contained in the work. Further transmission, reproduction, or presentation of this work is prohibited except with permission of the author(s).

Language dc:language.iso

eng

Handle dc:identifier.uri

https://hdl.handle.net/10657/9387

OAI identifier oai:identifier

oai:uh-ir.tdl.org:10657/9387