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Abstract
The biogenesis and assembly of the ribosome involves a coordinated cascade of events including rRNA processing, folding, and post-transcriptional modifications of rRNA along with the association of ribosomal proteins (r-proteins). Unraveling the pathways and dynamics of these complex structural processes is a significant challenge. While conventional biophysical techniques such as nuclear magnetic resonance (NMR), X-ray crystallography and cryo-electron microscopy (cryo-EM) provide high-resolution information, these methods are not ideally suited to characterize transient and heterogeneous ribosome assembly intermediate structures. Here mass spectrometry-based approaches are being used to gain insights into the composition and structural organization of ribosomes and ribosome assembly particles in vivo, particularly those particles that result from perturbations (e.g. deletion of assembly factors, antibiotics).
15N-labeling and data-dependent LC-MS/MS were used to characterize the proteins associated with pre-30S complexes from E. coli RimM and RbfA deletion strains. RimM and RbfA are ribosome assembly factors implicated in the maturation of the small 30S subunit in bacteria. The precise roles of these assembly factors in 30S subunit assembly are unclear. Along with in vivo x-ray footprinting and mass spectrometry data, detailed molecular mechanisms how RimM and RbfA facilitate maturation of the 30S subunit in vivo were uncovered.
Although relative quantitation of proteins by 15N-labeling and LC-MS/MS provides information on the differential expression of proteins in normal and perturbed samples, this approach is limited to comparing two samples at a time, labeling can be expensive and laborious, and not amenable to other multicellular organisms. The applicability of a label-free approach, LC-MSE for absolute "ribosome-centric" quantification of r-proteins was evaluated. Using an additional dimension of gas-phase separation through ion mobility and multiple endoproteinase digestion allow accurate and reproducible quantitation of proteins associated with mature ribosomes. The improved LC-MS E approach was then extended to characterizing proteins associated with different functional states of the ribosomes (free 30S, free 50S, 70S and polysomes). The actively translating ribosomes (polysomes) contain stoichiometric amounts of proteins consistent with their known stoichiometry within the complex. Significant heterogeneity was found with free subunits as they are composed of immature complexes and dissociated subunits from 70S. The stoichiometric measurements among the different classes of ribosomes showed very good run-to-run reproducibility and biological reproducibility with %CV less than 15% and 35%, respectively.
Finally, the in vivo assembly complexes formed in the presence of the antibiotic erythromycin was isolated and characterized. A strategy was devised to isolate and purify the erythromycin-induced 50S assembly particle in SK5665 cells grown in the presence of the antibiotic erythromycin. Quantitative analysis of the proteins associated with the Δ50S particles suggests a heterogeneous collection of 50S intermediates with different subsets and varying amounts of proteins. The amounts of the assembly factors, SrmB and DbpA, detected in the Δ50S particle indicate that the Δ50S particle is immature and is a late assembly intermediate.
