Genome-Wide Quantitation of Protein Synthesis Rates in Bacteria
Author(s)Johnson, Grace E.; Li, Gene-Wei
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Bacteria produce different amounts of their proteins in response to different conditions. The ability to accurately quantitate the rates of protein synthesis across the genome is an important step toward understanding both underlying regulation and bacterial physiology at a systems level. Ribosome profiling (deep sequencing of ribosome-protected mRNA fragments) enables accurate and high-throughput measurement of such synthesis rates. Ribosomes protect RNAs from nuclease digestion; thus, by collecting and sequencing protected footprints, one can obtain information on the position of every ribosome at the time of cell collection. Assuming ribosomes go on to translate full-length proteins, the density of ribosomes across an ORF can be used to determine protein synthesis rates. Here we outline a step-by-step protocol and discuss the steps where variability and bias may be introduced, including ways to minimize it.
DepartmentMassachusetts Institute of Technology. Department of Biology
Methods in enzymology
Johnson, Grace E. and Gene-Wei Li. “Genome-Wide Quantitation of Protein Synthesis Rates in Bacteria.” Methods in enzymology 612 (2018): 225-249 © 2018 The Author(s)
Author's final manuscript