User:Ddoerflinger/sandbox

I plan on writing more specific information regarding Ribosome profiling (RiboSeq) and adding more information since the the only information is based on the protocol, besides the lead.

These are the sources I plan on using:

1. Andreev, D. E., O’Connor, P. B. F., Loughran, G., Dmitriev, S. E., Baranov, P. V., & Shatsky, I. N. (2017). Insights into the Mechanisms of Eukaryotic Translation Gained with Ribosome Profiling. Nucleic Acids Research, 45(2), 513–526. https://doi.org/10.1093/nar/gkw1002
2. Buskirk, A. R., Green, R., & Green, R. (2017). Ribosome pausing , arrest and rescue in bacteria and eukaryotes. Philosophical Transactions of the Royal Society of London B: Biological Sciences, 372. https://doi.org/10.1098/rstb.2016.0183
3. Ingolia, N. T. (2014). Ribosome profiling: new views of translation, from single codons to genome scale. Nature Reviews. Genetics, 15(3), 205–13. https://doi.org/10.1038/nrg3645
4. Michel, A. M., & Baranov, P. V. (2013). Ribosome profiling: A Hi-Def monitor for protein synthesis at the genome-wide scale. Wiley Interdisciplinary Reviews: RNA, 4(5), 473–490. https://doi.org/10.1002/wrna.1172

Uses

There are three main uses of ribosome profiling: identifying translated mRNA regions, observing how nascent peptides are folded, and measuring the amount of specific proteins that are synthesized.

Identifying Translated mRNA Regions

By using specific drugs, ribosome profiling can identify either initiating regions of mRNA or elongating regions. Initiating regions can be detected by adding harringtonine or lactidomycin to prevent any further initiation. This allows the starting codon of the mRNAs throughout the cell lysate to be analyzed, which has been used to determine non-AUG sequences that do initiate translation. The other elongating regions can be detected by adding antibiotics like cyclohexamide that inhibit translocation, chloramphenical that inhibits transfer of peptides within the ribosome, or non-drug means like thermal freezing. These elongation freezing methods allow for the kinetics of translation to be analyzed. Since multiple ribosomes can translate a single mRNA molecule to speed up the translation process, RiboSeq demonstrates the protein coding regions within the mRNA and how quickly this is done depending on the mRNA being sequenced. This also allows for ribosome profiling to show pause sites within the transcriptome at specific codons. These sites of slow or paused translation are demonstrated by an increase in ribosome density and these pauses can link specific proteins with their roles within the cell.

Peptide Folding

Coupling ribosome profiling with ChIP can elucidate how and when newly synthesized proteins are folded. Using the footprints provided by Ribo-Seq, specific ribosomes associated with factors, like chaperones, can be purified. Pausing the ribosome at specific time points, allowing it to translate a polypeptide over time, and exposing the different points to a chaperone and precipitating out using ChIP purifies these samples and can show at which point in time the peptide is being folded.

Measuring Protein Synthesis

Ribo-Seq can also be used to measure protein synthesis and its regulators. This can be done by initially disrupting proteins that bind to RNA and using ribosome profiling to measure the difference in translation. These disrupted mRNAs can be associated with proteins, whose binding sites have already been mapped on RNA, to indicate regulation.

peer review

It is well organized and has a logical flow. It is helpful and supports the article. Additionally, it is easy to understand. One suggestion is that the addition of some "pros" and "cons" may improve the article.

Peer Review #2

This looks like a useful section that will add to the current Ribo-seq page. I would recommend breaking this "Uses" section into subsections; breaking up the information will make it easier for people to find specific points of interest. I think some of the sentences can be shortened to be more concise, otherwise well done.

Peer Review #3

I think you did a great job synthesizing this information. I would agree reviewer #2 that it may be helpful to break up the information into multiple sections. I also think it would be helpful to link the page to ChIP and Ribo-Seq pages to help readers who may not readily know what these concepts are.