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| Status |
Public on Feb 08, 2019 |
| Title |
A systematically-revised ribosome profiling protocol for bacteria reveals translational pauses at single-codon resolution |
| Organism |
Escherichia coli str. K-12 substr. MG1655 |
| Experiment type |
Other
|
| Summary |
In eukaryotes, ribosome profiling provides insight into the mechanism of protein synthesis at the codon level. In bacteria, however, the method has been more problematic and no consensus has emerged for how to best prepare profiling samples. Here, we identify the sources of these problems and describe new solutions for arresting translation and harvesting cells in order to overcome them. These improvements remove confounding artifacts and improve the resolution to allow analyses of ribosome behavior at the codon level. With a clearer view of the translational landscape in vivo, we observe that filtering cultures leads to translational pauses at serine and glycine codons through the reduction of tRNA aminoacylation levels. This observation illustrates how bacterial ribosome profiling studies can yield insight into the mechanism of protein synthesis at the codon level and how these mechanisms are regulated in response to changes in the physiology of the cell.
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| |
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| Overall design |
Ribosome profiling of E. coli MG1655 using a variety of lysis buffers and cell harvesting conditions
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| |
|
| Contributor(s) |
Buskirk AR |
| Citation(s) |
30724162, 38443396 |
| |
| Submission date |
Aug 27, 2018 |
| Last update date |
Mar 13, 2024 |
| Contact name |
Allen R Buskirk |
| E-mail(s) |
[email protected]
|
| Organization name |
Johns Hopkins University School of Medicine
|
| Department |
Molecular Biology and Genetics
|
| Street address |
725 N. Wolfe St
|
| City |
Baltimore |
| State/province |
MD |
| ZIP/Postal code |
21205 |
| Country |
USA |
| |
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| Platforms (1) |
| GPL18956 |
Illumina HiSeq 2500 (Escherichia coli str. K-12 substr. MG1655) |
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| Samples (10)
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| Relations |
| BioProject |
PRJNA488109 |
| SRA |
SRP158945 |
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