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PMID:22153074

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Contents

Citation

Oh, E, Becker, AH, Sandikci, A, Huber, D, Chaba, R, Gloge, F, Nichols, RJ, Typas, A, Gross, CA, Kramer, G, Weissman, JS and Bukau, B (2011) Selective ribosome profiling reveals the cotranslational chaperone action of trigger factor in vivo. Cell 147:1295-308

Abstract

As nascent polypeptides exit ribosomes, they are engaged by a series of processing, targeting, and folding factors. Here, we present a selective ribosome profiling strategy that enables global monitoring of when these factors engage polypeptides in the complex cellular environment. Studies of the Escherichia coli chaperone trigger factor (TF) reveal that, though TF can interact with many polypeptides, β-barrel outer-membrane proteins are the most prominent substrates. Loss of TF leads to broad outer-membrane defects and premature, cotranslational protein translocation. Whereas in vitro studies suggested that TF is prebound to ribosomes waiting for polypeptides to emerge from the exit channel, we find that in vivo TF engages ribosomes only after ~100 amino acids are translated. Moreover, excess TF interferes with cotranslational removal of the N-terminal formyl methionine. Our studies support a triaging model in which proper protein biogenesis relies on the fine-tuned, sequential engagement of processing, targeting, and folding factors.

Links

PubMed PMC3277850 Online version:10.1016/j.cell.2011.10.044

Keywords

Cytoplasm/chemistry; Escherichia coli/cytology; Escherichia coli/metabolism; Escherichia coli Proteins/metabolism; Membrane Proteins/chemistry; Membrane Proteins/metabolism; Molecular Chaperones/metabolism; Molecular Sequence Data; Peptidylprolyl Isomerase/metabolism; Protein Biosynthesis; Protein Transport; Ribosomes/metabolism

Significance

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  • Extended Ribosome Profiling to prokaryotes to enable the analysis of both translation and co-translational processes (such as Trigger Factor) that promote maturation of nascent polypeptides.

Useful Materials and Methods

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  • Four distinct steps (1) generation of cell extracts, in which ribosomes have been halted along the mRNA that they are translating; (2) treatment of polysomes with nuclease to remove regions of the message not protected by the ribosomes; (3) conversion of these RNA fragments into double stranded DNA copies and (4) analysis of these fragments by high throughput sequencing.

EcoliWiki data manipulation

  • Non-normalized wiggle files were sent from Eugene Oh; three replicates of dithiobis succinimidyl propionate (DSP) affinity purified (AP) for the forward strand, three replicates of DSP AP reverse strand, three replicates of the total ribosome profile for the forward strand, three replicates for the total profile of the reverse strand, one replicate each of 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide (EDC) AP forward and reverse strand, and one replicate each of EDC total ribosome profile forward and reverse strand. Replicates data sets were normalized by dividing all read values by the highest read value within each data set. After normalization, replicates were then averaged together. EDC replicates were normalized in the same manner, but not averaged due to only one replicate.

Annotations

Gene product Qualifier GO ID GO term name Evidence Code with/from Aspect Notes Status

Data

Data provided by the authors: Ribosome Profiling non-normalized Wiggle files(.zip)

Strain/Organism Assay Analyte Variable Conditions Notes Links

BW2952

Ribosome Profiling

Ribosomes

dithiobis succinimidyl propionate (DSP) total and 1-ethyl 3-[3-dimethylaminopropyl]carbodiimide (EDC) total

2 crosslinkers, forward and reverse strands

BW2952

Ribosome Profiling

Trigger Factor (tig)

dithiobis succinimidyl propionate (DSP) affinity purified (AP), 1-ethyl 3-[3-dimethylaminopropyl]carbodiimide (EDC) AP

Ribosomes crosslinked to trigger factor were affinity purified

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See also

References

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