Abstract
Ribosome profiling provides information on the position of ribosomes on mRNA on a genomic scale. Although this information is often used to detect changes in gene expression under different conditions, it also has great potential for yielding insight into the mechanism and regulation of protein synthesis itself. First developed in yeast, ribosome profiling involves the isolation and sequencing of ribosome-protected mRNA fragments generated by nuclease treatment. Since the application of ribosome profiling in bacteria has been problematic, we report here a systematically optimized protocol for E. coli that we have used with success for other bacteria as well. Cells are harvested by flash-freezing cultures directly in liquid nitrogen. After lysis, translation is arrested by high magnesium concentration without the use of antibiotics. These improvements eliminate artifacts induced by harvesting cells by centrifugation or filtration and by use of chloramphenicol to arrest translation. These improvements are especially appropriate for studies where the exact position of the ribosome is critical, and not merely the number of ribosomes per message, such as studies aimed at monitoring differences in local elongation rates.
Highlights
11. Aluminum foilD. PCR primer Forward (100 nmol; not gel purified):
[Background] mRNAs are translated at varying efficiencies depending on properties inherent to the mRNA such as initiation rates, mRNA structure, and codon usage
First developed by Ingolia and Weissman in yeast (Ingolia et al, 2009), ribosome profiling utilizes nuclease digestion to degrade regions of mRNA that are not occupied by ribosomes, generating ribosome-protected mRNA fragments that are isolated, cloned, and sequenced
Summary
D. PCR primer Forward (100 nmol; not gel purified):. Agilent High Sensitivity DNA Kit (Bio Analyzer) (Agilent Technologies, catalog number: 5067-4626) 45. GlycoBlue (Invitrogen Life Technologies, catalog number: AM9516) 46. SYBR® Gold Nucleic Acid Gel Stain (Invitrogen Life Technologies, catalog number: S-11494) 47. 10% TBE Urea 1.0 mm (18 + 2) 30 μl gel (Bio-Rad, catalog number: 345-0089) (1 per experiment) 52. 10% TBE Native 1.0 mm (12 + 2) 45 μl gel (Bio-Rad, catalog number: 345-0051) (4-6 per experiment) 53. 50% Sucrose Gradient Buffer (see Recipes) 58. Analysis of sequenced ribosome footprints was done using a custom python pipeline found in the link below: https://github.com/greenlabjhmi/2018_Bacterial_Pipeline_riboseq
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