The molecular choreography of protein synthesis : single-molecule profiling of ribosome recording phenomena
Abstract/Contents
- Abstract
- Translation of proteins by the ribosome regulates gene expression; misregulation of translation is a leading cause of many diseases, including cancer and many genetic diseases. Currently, there is a general misconception that translation is a linear process with a single mechanistic pathway. However, translation likely involves multiple pathways and branchpoints, with the stochastic nature of biological processes allowing for different pathways to occur that are biased by the interaction of the ribosome with other translation factors. These multiple pathways and branchpoints are potential regulatory points, allowing gene expression to be tuned at the translational level by messenger RNA (mRNA) sequence and structure. Shine-Dalgarno sequences, mRNA hairpins and pseudoknots, as well as nascent peptide-ribosome interactions, are known to pause or stall the ribosome. These stimulatory elements may lead to kinetic branchpoints during elongation and induce recoding events, wherein the ribosome is shunted into alternative pathways that result in either changes in reading frame or the bypassing of a region of the mRNA. Here, I first present the development of single-molecule fluorescence methods and nanophotonic instrumentation (zero-mode waveguides, ZMWs) to allow direct profiling of translational rates of thousands of single ribosomes with codon resolution. Using these techniques, I investigated the dynamic mechanisms of two recoding events: the -1 frameshifting in the dnaX gene and the ribosome bypassing of a 50 nucleotide untranslated region in gene 60 of T4 phage. We observed multiple pathways induced by the stochastic interaction of the ribosome with the stimulatory elements; the ribosomes that undergo recoding in both frameshifting and bypassing are characterized by a pause in the rotated state. Such paused states allow unusual events in elongation and may be a central feature of translational control.
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 | Chen, Jin, (Researcher in cellular molecular pharmacology) |
|---|---|
| Associated with | Stanford University, Department of Applied Physics. |
| Primary advisor | Puglisi, Joseph D |
| Primary advisor | Quake, Stephen Ronald |
| Thesis advisor | Puglisi, Joseph D |
| Thesis advisor | Quake, Stephen Ronald |
| Thesis advisor | Levitt, Michael, 1947- |
| Advisor | Levitt, Michael, 1947- |
Subjects
| Genre | Theses |
|---|
Bibliographic information
| Statement of responsibility | Jin Chen. |
|---|---|
| Note | Submitted to the Department of Applied Physics. |
| Thesis | Thesis (Ph.D.)--Stanford University, 2015. |
| Location | electronic resource |
Access conditions
- Copyright
- © 2015 by Jin Chen
- License
- This work is licensed under a Creative Commons Attribution Non Commercial 3.0 Unported license (CC BY-NC).
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