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| Status |
Public on Jul 10, 2019 |
| Title |
Selective ribosome profiling to study interactions of translating ribosomes in yeast |
| Organism |
Saccharomyces cerevisiae |
| Experiment type |
Other
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| Summary |
A number of enzymes, targeting factors and chaperones engage ribosomes to support fundamental steps of nascent protein maturation, including enzymatic processing, membrane targeting and co-translational folding. The selective ribosome profiling (SeRP) method is a new tool for studying the co-translational activity of maturation factors that provides proteome-wide information on a factor’s nascent interactome, the onset and duration of binding and the mechanisms controlling factor engagement. SeRP is based on the combination of two ribosome-profiling (RP) experiments, sequencing the ribosome-protected mRNA fragments from all ribosomes (total translatome) and the ribosome subpopulation engaged by the factor of interest (factor-bound translatome). We provide a detailed SeRP protocol, exemplified for the yeast Hsp70 chaperone Ssb (stress 70 B), for studying factor interactions with nascent proteins that is readily adaptable to identifying nascent interactomes of other co-translationally acting eukaryotic factors. The protocol provides general guidance for experimental design and optimization, as well as detailed instructions for cell growth and harvest, the isolation of (factor-engaged) monosomes, the generation of a cDNA library and data analysis. Experience in biochemistry and RNA handling, as well as basic programing knowledge, is necessary to perform SeRP. Execution of a SeRP experiment takes 8–10 working days, and initial data analysis can be completed within 1–2 d. This protocol is an extension of the originally developed protocol describing SeRP in bacteria.
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| Overall design |
Method description. Control mixing experiment to show that no post-lysis binding occurs.
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| Contributor(s) |
Galmozzi CV, Merker D, Friedrich U, Döring K, Kramer G |
| Citation(s) |
31332354 |
| Submission date |
Nov 30, 2018 |
| Last update date |
Oct 09, 2019 |
| Contact name |
Kristina Rebekka Doering |
| Organization name |
ZMBH
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| Department |
University of Heidelberg
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| Lab |
AG Bukau
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| Street address |
Im Neuenheimer Feld 282
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| City |
Heidelberg |
| State/province |
Baden-Württemberg |
| ZIP/Postal code |
69120 |
| Country |
Germany |
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| Platforms (1) |
| GPL13821 |
Illumina HiSeq 2000 (Saccharomyces cerevisiae) |
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| Samples (8)
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| Relations |
| BioProject |
PRJNA507750 |
| SRA |
SRP171612 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE123166_RAW.tar |
355.7 Mb |
(http)(custom) |
TAR (of TXT) |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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