RNA transcript regulation studied at the single-molecule level
Abstract/Contents
- Abstract
- The synthesis and degradation of RNA is highly regulated in all living organisms to ensure proper gene expression. In E. coli, RNA synthesis is controlled by a wide variety of proteins that modulate the behavior of RNA polymerase in accordance with the needs of the cell. Once an RNA molecule is synthesized, its lifetime is set by the activity and substrate specificity of ribonucleases, essential enzymes that enable rapid changes in gene expression by preventing the accumulation of old transcripts. Here, I use optical tweezers to examine three RNA-motor proteins that play fundamental roles in the regulation of bacterial transcripts. First I present a single-molecule study of E. coli Rho factor, the helicase responsible for terminating transcription of 20-50% of all mRNA. I identify two separate RNA-binding states for Rho, establish that it advances 5′ to 3′ via a tethered-tracking mechanism, and present an overall model for its binding and translocation behavior. I then introduce a novel single-molecule assay for observing processive motion of helicases and exonucleases, and use this technique to examine two of the major enzymes responsible for degrading RNA in bacteria -- RNase R and polynucleotide phosphorylase. I find that individual RNase R enzymes exhibit sequence-dependent pausing behavior as they processively unwind hundreds of base pairs of dsRNA, and that polynucleotide phosphorylase moves 3′ to 5′ along a transcript in discrete 6-7 nt steps, a result consistent with an asymmetric inchworm mechanism of translocation. Overall, this work provides key mechanistic insights into the behaviors of three vital enzymes that modulate gene expression in bacteria via their interactions with RNA.
Description
| Type of resource | text |
|---|---|
| Form | electronic; electronic resource; remote |
| Extent | 1 online resource. |
| Publication date | 2014 |
| Issuance | monographic |
| Language | English |
Creators/Contributors
| Associated with | Koslover, Daniel Jacob |
|---|---|
| Associated with | Stanford University, Biophysics Program. |
| Primary advisor | Block, Steven M |
| Thesis advisor | Block, Steven M |
| Thesis advisor | Greenleaf, William James |
| Thesis advisor | Theriot, Julie |
| Advisor | Greenleaf, William James |
| Advisor | Theriot, Julie |
Subjects
| Genre | Theses |
|---|
Bibliographic information
| Statement of responsibility | Daniel Jacob Koslover. |
|---|---|
| Note | Submitted to the Program in Biophysics. |
| Thesis | Thesis (Ph.D.)--Stanford University, 2014. |
| Location | electronic resource |
Access conditions
- Copyright
- © 2014 by Daniel Jacob Koslover
- License
- This work is licensed under a Creative Commons Attribution Non Commercial 3.0 Unported license (CC BY-NC).
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