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Figure 1 | BMC Genomics

Figure 1

From: A comparative genomics study on the effect of individual amino acids on ribosome stalling

Figure 1

General description of the approach described in the study. (A) The major steps of the ribosomal profiling approach: 1) Cells are treated with cycloheximide, for example, to arrest translation; 2) Ribosomes are fixed and ribosome-protected RNA fragments are recovered; 3) After processing and reverse-transcription, these are sequenced, mapped and used to derive a ribosomal density profile. (B) An illustration of the ribosome and the exit tunnel during translation elongation. The sequence of codons upstream from the ribosomal A-site (shaded in gray) represents the amino acid sequence that occupies the exit tunnel while the codon at the P-site is being translated (depicted by pink circles). (C) The general steps of the approach described in this study: Ribo-seq and mRNA-seq profiles are normalized by the average gene coverage; new profiles are generated based on the ratio between ribo-seq reads and mRNA reads; normalized profiles with sparse coverage are filtered; peak positions in RD/mRNA are extracted; the codons USR of each peak is converted into amino acid sequence (denoted as AA) and each amino acid is analyzed based on its frequency in all USRs (see specific details in the Methods). (D) An example of ribo-seq, mRNA-seq and RD/mRNA profiles obtained from gene YAL012W in S. cerevisiae. The profiles were generated based on all S. cerevisiae datasets (see the Methods section: Merging all datasets of the organism into one aggregate). Positions along each profile represent the location of the ribosomal A-site. The first 20 codons (marked by a dashed brown frame) are excluded from the analysis (details in the Methods section: Data filtering). The 31 codons upstream from the peak are the Upstream Stalling Region of codons corresponding to the amino acid sequence in the exit tunnel.

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