Single-molecule studies of real protein synthesis
Abstract/Contents
- Abstract
- Translation, the last step in the process of cellular gene expression, is essential to life in all of its forms. At the center of translation is the ribosome, a multi-megadalton assembly of highly structured RNA and numerous proteins that together form a molecular-machine capable of both reading the genetic code and catalyzing the formation of the peptide bonds that connect the amino-acid building blocks of proteins. The ribosomal exit tunnel is the last point of interaction between the newly synthesized protein and the cellular gene-expression machinery and was for years thought to play only a passive role in the synthesis of protein, serving as nothing more than an inert conduit through which the peptide passes on its way into the cytosol. However, over the last several years an increasing body of evidence has suggested an active role for the tunnel in the control of expression of certain peptide sequences. Among these is SecM, a bacterial secretion monitor capable of stalling translation elongation on the prokaryotic ribosome in a co-factor independent manner. Extensive biochemical and cryo-EM structural studies have demonstrated that the SecM nascent chain stalls the ribosome by specifically interacting with the 50S subunit exit tunnel to inhibit peptide bond formation. However, how the SecM sequence changes translation dynamics prior to stalling and whether there are other necessary interactions with the ribosome not captured in the final stalled state, remains unclear. Using single-molecule fluorescence techniques combined with newly developed 'complete' in-vitro translation systems I monitored ribosome conformation and composition over the course of translation of a number of complex mRNA sequences. I demonstrate that the mechanism of SecM stalling involves a cascade of precisely timed peptide / tunnel interactions. In addition, the existence of a heretofore unknown family of E.coli stall sequences was conclusively demonstrated, and it was shown that some of the interactions involved in SecM-induced stalling contribute also to ribosomal stalling during translation of this new family of stall sequences. These results underscore the roles that the 50S subunit exit tunnel and nascent chain play in controlling translation and gene expression, while demonstrating the power and generality of single-molecule techniques to the discovery of previously unknown molecular phenomena and their mechanisms of action.
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 | Kornberg, Guy Joseph Lorch |
|---|---|
| Associated with | Stanford University, Department of Structural Biology. |
| Primary advisor | Puglisi, Joseph D |
| Thesis advisor | Puglisi, Joseph D |
| Thesis advisor | Levitt, Michael, 1947- |
| Thesis advisor | Sarnow, P. (Peter) |
| Advisor | Levitt, Michael, 1947- |
| Advisor | Sarnow, P. (Peter) |
Subjects
| Genre | Theses |
|---|
Bibliographic information
| Statement of responsibility | Guy Joseph Lorch Kornberg. |
|---|---|
| Note | Submitted to the Department of Structural Biology. |
| Thesis | Thesis (Ph.D.)--Stanford University, 2014. |
| Location | electronic resource |
Access conditions
- Copyright
- © 2014 by Guy Joseph Lorch Kornberg
- License
- This work is licensed under a Creative Commons Attribution Non Commercial 3.0 Unported license (CC BY-NC).
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