Isolation of bacterial compartments to track movement of protein synthesis factors

Aminoacyl-tRNA synthetases (AARSs) comprise an enzyme family that generates and maintains pools of aminoacylated tRNAs, which serve as essential substrates for protein synthesis. Many protein synthesis factors, including tRNA and AARSs also have non-canonical functions. Particularly in mammalian cells, alternate functions of AARSs have been associated with re-distribution in the cell to sites that are removed from translation. Sub-fractionation methods for E. coli were designed and optimized to carefully investigate re-localization of bacterial AARSs and tRNA that might aid in conferring alternate activities. Cell fractionation included isolation of the cytoplasm, periplasm, membrane, outer membrane vesicles, and extracellular media. Specific endogenous proteins and RNAs were probed respectively within each fraction via Western blots using antibodies and by Northern blots with primers to unique regions of the nucleic acid.