Simultaneous dual-beam optical trap and sm-fret probing long-range tertiary interactions in tpp riboswitch
Abstract/Contents
- Abstract
- RNAs are ubiquitous in virtually all biological processes and its function is based on their 3D structures. From sequence to 3D structure, its ability to carry genetic information and function like proteins shows remnants of a RNA world (Breaker, 2012). Riboswitches are evidence of a RNA world as they are cis-regulatory elements formed in mRNA that can modify gene expression in response to environmental cues. A well-known example is the thiamine pyrophosphate (TPP) riboswitch, whose specificity for TPP is dependent upon conformational changes that take place within its ligand-binding aptamer region. Here, the role of tertiary interactions in ligand binding is studied at the single-molecule level by combined force spectroscopy and Fӧrster resonance energy transfer (smFRET), using a dual-beam optical trapping apparatus equipped for simultaneous, spatially coincident smFRET. Detailed calibrations and optimization of the "Force-FRET" technique are performed using several well-characterized DNA hairpins by force spectroscopy. Extending this approach, we directly probe secondary and tertiary structural changes during TPP aptamer folding, including key events associated with ligand binding. The concurrent transitions observed in smFRET signals and RNA length changes revealed differences in helix arm orientation between two previously-identified ligand-binding states that were undetectable by force spectroscopy alone. With our Force-FRET technique, we can resolve the structural details of the weaker binding state when fully bind to TPP, and identify the mechanism behind its inability to form the tertiary interaction that completes the TPP binding process. This powerful single-molecule technique enhances our understanding of how tertiary interactions play a crucial role in stabilizing global riboswitch structure and, ultimately, in increasing ligand specificity. Further investigation of these multidimensional energy landscape for RNA folding require development of techniques, such as Force-FRET, that can probe this landscape with more than one reaction coordinate. This development enables us to ask more complicated questions such as coupling transcription-translation and observing both processivity and conformational changes of these proteins.
Description
| Type of resource | text |
|---|---|
| Form | electronic; electronic resource; remote |
| Extent | 1 online resource. |
| Publication date | 2015 |
| Issuance | monographic |
| Language | English |
Creators/Contributors
| Associated with | Duesterberg, Van Khanh Nguyen |
|---|---|
| Associated with | Stanford University, Department of Biophysics. |
| Primary advisor | Block, Steven M |
| Thesis advisor | Block, Steven M |
| Thesis advisor | Das, Rhiju |
| Thesis advisor | Dunn, Alexander Robert |
| Thesis advisor | Riedel-Kruse, Hans |
| Advisor | Das, Rhiju |
| Advisor | Dunn, Alexander Robert |
| Advisor | Riedel-Kruse, Hans |
Subjects
| Genre | Theses |
|---|
Bibliographic information
| Statement of responsibility | Van Khanh Nguyen Duesterberg. |
|---|---|
| Note | Submitted to the Department of Biophysics. |
| Thesis | Thesis (Ph.D.)--Stanford University, 2015. |
| Location | electronic resource |
Access conditions
- Copyright
- © 2015 by Van Khanh Nguyen Duesterberg
- License
- This work is licensed under a Creative Commons Attribution Non Commercial 3.0 Unported license (CC BY-NC).
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