A combined quantitative mass spectrometry and electron microscopy analysis of ribosomal 30S subunit assembly in E. coli

摘要

The proteins in cells are made by complex organelles called ribosomes. These organelles are made of two subunits the small ribosomal subunit, which reads the messenger RNA that contains the genetic code for the protein, and the large ribosomal subunit, which links amino acids together to form a protein. But how are the ribosomes themselves—which contain several ribosomal RNA molecules and dozens of ribosomal proteins—put together? Various aspects of the assembly of ribosomes have been studied in the test tube, but the complexity of the assembly process means there is little data from experiments performed on living cells. Now Sashital et al. have used a combination of two techniques—mass spectrometry and electron microscopy—to study the assembly of ribosomes in living Escherichia coli cells. Mass spectrometry measures the relative amounts of the different ribosomal proteins in each sample, while electron microscopy provides information on the shape of the ribosome, including the shape of some of the intermediate structures formed during the assembly process. Sashital et al. analyzed the composition and structure of the small ribosomal subunits in wild type E. coli, and also in mutant E. coli cells in which the genes for various proteins thought to be involved in the assembly process had been deleted. These experiments revealed that a protein called RimP had a key role in stabilizing an important central structure called a pseudoknot. The approach developed by Sashital et al. should be able to reveal other details about the assembly of ribosomes, and also about other macromolecular complexes that are found inside the cells.