Project description:We present a ribosome profiling data set from S. cerevisiae as part of a larger study that models the elongation rate along transcripts as a function of local sequence neighborhoods. We then apply this model to design coding sequences that are optimized for translational output.
Project description:Synonymous codon choice can have dramatic effects on ribosome speed and protein expression. Ribosome profiling experiments have underscored that ribosomes do not move uniformly along mRNAs. Here, we have modeled this variation in translation elongation by using a feed-forward neural network to predict the ribosome density at each codon as a function of its sequence neighborhood. Our approach revealed sequence features affecting translation elongation and characterized large technical biases in ribosome profiling. We applied our model to design synonymous variants of a fluorescent protein spanning the range of translation speeds predicted with our model. Levels of the fluorescent protein in budding yeast closely tracked the predicted translation speeds across their full range. We therefore demonstrate that our model captures information determining translation dynamics in vivo; that this information can be harnessed to design coding sequences; and that control of translation elongation alone is sufficient to produce large quantitative differences in protein output.
Project description:E.coli K-12 W3110 was grown in LB medium and harvested at each time point. And time-series microarray experiments were performed based on Sequential Design. In Sequential Design, the control sample is set to closest previous time point so that adjacent time points are compared directly. Combining with data from reference design, more accurate and reliable expression series could be collected. Keywords: timecourse
Project description:DNA damage activates a robust transcriptional stress response, but much less is known about how DNA damage impacts translation. The advent of genome editing with Cas9 has intensified interest in understanding cellular responses to DNA damage. Here, we find that DNA double-strand breaks (DSBs), including those induced by Cas9, trigger the loss of ribosomal protein RPS27A from ribosomes via p53-independent proteasomal degradation. Comparisons of Cas9 and dCas9 ribosome profiling and mRNA-seq experiments reveal a global translational response to DSBs that precedes changes in transcript abundance. Our results demonstrate that even a single DSB can lead to altered translational output and ribosome remodeling, suggesting caution in interpreting cellular phenotypes measured immediately after genome editing.
Project description:Ribosome profiling is a powerful method for globally assessing the activity of ribosomes in a cell. Despite its application in many organisms, ribosome profiling studies in bacteria have struggled to obtain the resolution necessary to precisely define translational pauses. Here we report improvements that yield much higher resolution in E. coli profiling data, enabling us to more accurately assess ribosome pausing and refine earlier studies of the impact of polyproline motifs on elongation. We comprehensively characterize pausing at proline-rich motifs in the absence of elongation factor EFP. We find that only a small fraction of genes with strong pausing motifs have reduced ribosome density downstream and identify features that explain this phenomenon. These features allow us to predict which proteins likely have reduced output in the efp knockout strain. Ribosome profiling of E. coli MG1655 and mutants lacking EFP or its three modifiying enzymes
Project description:The rate of protein synthesis varies according to the mRNA sequence in ways that affect gene expression. Global analysis of translational pausing is now possible with ribosome profiling. Here, we revisit an earlier report that Shine-Dalgarno sequences are the major determinant of translational pausing in bacteria. Using refinements in the profiling method as well as biochemical assays, we find that SD motifs have little (if any) effect on elongation rates. We argue that earlier evidence of pausing arose from two factors. First, in previous analyses, pauses at Gly codons were difficult to distinguish from pauses at SD motifs. Second, and more importantly, the initial study preferentially isolated long ribosome-protected mRNA fragments that are enriched in SD motifs. These findings clarify the landscape of translational pausing in bacteria as observed by ribosome profiling. Ribosome profiling (three replicates) and RNAseq (two replicates) of E. coli MG1655
Project description:Ribosome profiling is a powerful method for globally assessing the activity of ribosomes in a cell. Despite its application in many organisms, ribosome profiling studies in bacteria have struggled to obtain the resolution necessary to precisely define translational pauses. Here we report improvements that yield much higher resolution in E. coli profiling data, enabling us to more accurately assess ribosome pausing and refine earlier studies of the impact of polyproline motifs on elongation. We comprehensively characterize pausing at proline-rich motifs in the absence of elongation factor EFP. We find that only a small fraction of genes with strong pausing motifs have reduced ribosome density downstream and identify features that explain this phenomenon. These features allow us to predict which proteins likely have reduced output in the efp knockout strain.
Project description:Purpose: The aim is to analyze the translational kinetics of the basal and LPS-stimulated conditions of RAW264 macrophages focusing on ribosome density Method: RAW264 macrophages were cultured at 3.0 x 10^5 cells/ml in media (DMEM, 2mM Glutamine, 10% FBS, 100 units penicillin and 100µg streptomycin/mL) 24 hours prior to harvest. It was confirmed that the confluency of cells never surpassed 80 ~ 90%. Cell were harvested in their basal state or LPS stimulated condition (100 ng/ml for 30 min) and ribosome profiling (Ribo-Seq) and high-throughput mRNA sequencing (mRNA-Seq) were conducted. Ribosome protected fragments (RPF) were sequenced and the density was normalized by mRNA abundance. Result and conclusion: We discovered where in the ribosome translational arrest ocurs and how this happens. Translational stall was striking at A-site and P-site of ribosomal complex. The other arrest site was observed 3 to 5 residues away from the peptidyl transfer center of the exit tunnel, in which ribosomal density and hydrophobicity showed a significant negative correlation. mRNA and RPF of RAW264 macrophages were deep-sequenced with two independent biological replicates by Ion PGM sequencer