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1. RiboA: a web application to identify ribosome A-site locations in ribosome profiling data

   https://doi.org/10.1186/s12859-021-04068-w  

   Shao, Danying ; Ahmed, Nabeel ; Soni, Nishant ; O’Brien, Edward P. ( December 2021 , BMC Bioinformatics)

   Abstract Background Translation is a fundamental process in gene expression. Ribosome profiling is a method that enables the study of transcriptome-wide translation. A fundamental, technical challenge in analyzing Ribo-Seq data is identifying the A-site location on ribosome-protected mRNA fragments. Identification of the A-site is essential as it is at this location on the ribosome where a codon is translated into an amino acid. Incorrect assignment of a read to the A-site can lead to lower signal-to-noise ratio and loss of correlations necessary to understand the molecular factors influencing translation. Therefore, an easy-to-use and accurate analysis tool is needed to accurately identify the A-site locations. Results We present RiboA, a web application that identifies the most accurate A-site location on a ribosome-protected mRNA fragment and generates the A-site read density profiles. It uses an Integer Programming method that reflects the biological fact that the A-site of actively translating ribosomes is generally located between the second codon and stop codon of a transcript, and utilizes a wide range of mRNA fragment sizes in and around the coding sequence (CDS). The web application is containerized with Docker, and it can be easily ported across platforms. Conclusions The Integer Programming method that RiboA utilizesmore »is the most accurate in identifying the A-site on Ribo-Seq mRNA fragments compared to other methods. RiboA makes it easier for the community to use this method via a user-friendly and portable web application. In addition, RiboA supports reproducible analyses by tracking all the input datasets and parameters, and it provides enhanced visualization to facilitate scientific exploration. RiboA is available as a web service at https://a-site.vmhost.psu.edu/ . The code is publicly available at https://github.com/obrien-lab/aip_web_docker under the MIT license.« less
2. In vitro ribosome synthesis and evolution through ribosome display

   https://doi.org/10.1038/s41467-020-14705-2  

   Hammerling, Michael J. ; Fritz, Brian R. ; Yoesep, Danielle J. ; Kim, Do Soon ; Carlson, Erik D. ; Jewett, Michael C. ( February 2020 , Nature Communications)

   Directed evolution of the ribosome for expanded substrate incorporation and novel functions is challenging because the requirement of cell viability limits the mutations that can be made. Here we address this challenge by combining cell-free synthesis and assembly of translationally competent ribosomes with ribosome display to develop a fully in vitro methodology for ribosome synthesis and evolution (called RISE). We validate the RISE method by selecting active genotypes from a ~1.7 × 10⁷member library of ribosomal RNA (rRNA) variants, as well as identifying mutant ribosomes resistant to the antibiotic clindamycin from a library of ~4 × 10³rRNA variants. We further demonstrate the prevalence of positive epistasis in resistant genotypes, highlighting the importance of such interactions in selecting for new function. We anticipate that RISE will facilitate understanding of molecular translation and enable selection of ribosomes with altered properties.
3. Insights into the ribosome function from the structures of non-arrested ribosome–nascent chain complexes

   https://doi.org/10.1038/s41557-022-01073-1  

   Syroegin, Egor A. ; Aleksandrova, Elena V. ; Polikanov, Yury S. ( January 2023 , Nature Chemistry)
4. Foldamers wave to the ribosome

   https://doi.org/10.1038/s41557-018-0036-5  

   Schepartz, Alanna ( April 2018 , Nature Chemistry)
5. Temperature-dependent radiation sensitivity and order of 70S ribosome crystals

   https://doi.org/10.1107/S1399004714017672  

   Warkentin, Matthew ; Hopkins, Jesse B. ; Haber, Jonah B. ; Blaha, Gregor ; Thorne, Robert E. ( November 2014 , Acta Crystallographica Section D Biological Crystallography)

   All evidence to date indicates that at T = 100 K all protein crystals exhibit comparable sensitivity to X-ray damage when quantified using global metrics such as change in scaling B factor or integrated intensity versus dose. This is consistent with observations in cryo-electron microscopy, and results because nearly all diffusive motions of protein and solvent, including motions induced by radiation damage, are frozen out. But how do the sensitivities of different proteins compare at room temperature, where radiation-induced radicals are free to diffuse and protein and lattice structures are free to relax in response to local damage? It might be expected that a large complex with extensive conformational degrees of freedom would be more radiation sensitive than a small, compact globular protein. As a test case, the radiation sensitivity of 70S ribosome crystals has been examined. At T = 100 and 300 K, the half doses are 64 MGy (at 3 Å resolution) and 150 kGy (at 5 Å resolution), respectively. The maximum tolerable dose in a crystallography experiment depends upon the initial or desired resolution. When differences in initial data-set resolution are accounted for, the former half dose is roughly consistent with that for model proteins, and the 100/300 K half-dose ratio is roughly amore »factor of ten larger. 70S ribosome crystals exhibit substantially increased resolution at 100 K relative to 300 K owing to cooling-induced ordering and not to reduced radiation sensitivity and slower radiation damage.« less