#Ribosome profiling viewer how to

Although codon usage, amino acid sequence, and mRNA structure have each been shown to affect local elongation rates, our current understanding of these various features is insufficient to predict their effects on single genes, let alone across the genome. Just as elongation can reshape mRNA structures, the converse is also true: strong mRNA structures can pause ribosomes and trigger rescue pathways associated with ribosome stalling and arrest ( Doma and Parker, 2006). coli SecM peptide uses stalling as a key feature in an elegant genetic switch in which stalling leads to changes in the local mRNA structure that promote translation of the downstream secA gene ( Nakatogawa and Ito, 2002). Interactions between side chains and the exit tunnel can rearrange nucleotides within the peptidyl-transferase center, locking the ribosome in an inactive conformation ( Ito and Chiba, 2013). The amino acid sequence of the nascent polypeptide can also alter elongation rates. In a similar manner, rare codons in structural genes have been implicated in fine-tuning translational rates in order to favor proper protein folding ( Kim et al., 2015 Kimchi-Sarfaty et al., 2007 Komar, 2009 Zhou et al., 2013). Stretches of codons in leader peptides that are translated slowly under starvation conditions, for example, regulate the transcription of downstream biosynthesis genes (e.g. These variations can have dramatic effects on gene expression. Local elongation rates vary considerably during protein synthesis depending on the codon, amino acid sequence, and mRNA structure. This observation illustrates how bacterial ribosome profiling studies can yield insight into the mechanism of protein synthesis at the codon level and how these mechanisms are regulated in response to changes in the physiology of the cell. With a clearer view of the translational landscape in vivo, we observe that filtering cultures leads to translational pauses at serine and glycine codons through the reduction of tRNA aminoacylation levels. These improvements remove confounding artifacts and improve the resolution to allow analyses of ribosome behavior at the codon level. Here, we identify the sources of these problems and describe new solutions for arresting translation and harvesting cells in order to overcome them.

#Ribosome profiling viewer how to

In bacteria, however, the method has been more problematic and no consensus has emerged for how to best prepare profiling samples. In eukaryotes, ribosome profiling provides insight into the mechanism of protein synthesis at the codon level.