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| Status |
Public on Feb 25, 2019 |
| Title |
Assembly and functionality of the ribosome with tethered subunits |
| Organism |
Escherichia coli |
| Experiment type |
Other
Expression profiling by high throughput sequencing
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| Summary |
Ribo-T is an engineered ribosome whose small and large subunits are tethered together by insertion of circularly permutated 23S rRNA into 16S rRNA [Orelle, C., Carlson, E. D., Szal, T., Florin, T., Jewett, M. C., Mankin, A.S. Protein synthesis by ribosomes with tethered subunits. Nature 524, 119-124 (2015)]. Whether the growth defects are related to problems with Ribo-T functionality is unclear. The translation in Ribo-T cells was examined by ribosome profiling (Ribo-seq) and RNA sequencing (RNA-seq). Besides the control cells carrying dissociable WT ribosome (SQ171), RIBO-T cells are the original SQ171 strain transformed with pRibo-T plasmid and cured of the plasmid carrying wt rRNA that was evolved to have improved growth characteristics. The Ribo-T strain carries two mutations: a nonsense mutation in the Leu22 codon of the ybeX gene and a missense mutation in codon 549 of the rpsA gene encoding ribosomal protein S1. Analysis of Ribo-T performance by ribosome profiling showed a notably higher ribosome occupancy of the start codons and somewhat increased occupancy at the stop codons of many genes, suggesting that subunit tethering specifically impairs the initiation and termination stages of translation.
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| Overall design |
Ribosome profiling and RNA-seq was performed on Escherichia coli Ribo-T (a derivative of the strain SQ171, 2 biological replicates) and compared to a SQ171 control cells carrying WT ribosomes (2 biological replicates).
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| Contributor(s) |
Aleksashin NA, Leppik M, Hockenberry AJ, Klepacki D, Vázquez-Laslop N, Jewett MC, Remme J, Mankin AS |
| Citation(s) |
30804338 |
| |
| Submission date |
Sep 04, 2018 |
| Last update date |
Mar 06, 2019 |
| Contact name |
Adam John Hockenberry |
| E-mail(s) |
adam.hockenberry@utexas.edu
|
| Organization name |
The University of Texas at Austin
|
| Department |
Department of Integrative Biology
|
| Lab |
Institute for Cellular and Molecular Biology
|
| Street address |
2500 Speedway, A4800, MBB 3.232
|
| City |
Austin |
| State/province |
TX |
| ZIP/Postal code |
78712 |
| Country |
USA |
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| Platforms (1) |
| GPL21222 |
Illumina NextSeq 500 (Escherichia coli) |
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| Samples (8)
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| Relations |
| BioProject |
PRJNA489275 |
| SRA |
SRP159555 |
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