The Assembly Landscape of the 30S Ribosomal Subunit Monitored with Mass Spectrometry PDF Download

Author: Megan Wright Trevathan
Publisher:
ISBN:
Category : Chemical kinetics
Languages : en
Pages : 626

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Book Description
The architecture of the bacterial ribosome is now known at atomic resolution. A similarly detailed picture of how the rRNA and protein components assemble into this structure is still being drawn. In this work, a new method is developed to probe the mechanism of assembly of the E. coli 30S ribosomal subunit. The method, which measures RNA-protein binding kinetics in vitro with isotopic labeling and quantitative protein mass spectrometry, monitors simultaneously the binding kinetics of individual proteins to the assembling 30S subunit and thus reports on the kinetics of assembling the many parts of the structure. The method reveals that the proteins generally bind in single kinetic phases. The events limiting protein binding are a mix of bimolecular (rRNA-protein binding) and unimolecular (folding) transitions. In no case is folding dramatically slower than binding. 30S assembly is understood to be limited by an energy-dependent conformational change of a single intermediate. Data from this study indicate that assembling 30S subunits populate multiple intermediates; three are proposed, each of which corresponds to a particular region of the 30S structure. One follows initial assembly of the 5 ' domain and some assembly in the central domain. The second follows further 5' domain assembly. The third follows assembly of the central domain. Much of the 3 ' domain, along with the mRNA decoding site at the junction of the three domains, assembles after the last intermediate. It appears that no single transition is particularly energy-dependent; rather, the many steps of assembly have similar activation energies. Mg2+ is needed in high concentrations (at least 10 mM) for complete, efficient 30S assembly. Here it is shown that high [Mg 2+ ] is required for the later steps, in which the 3' domain and mRNA decoding site are assembled. The earlier steps of assembly, however, are slowed down by high [Mg2+ ]. A study of the folding of another RNA, the Tetrahymena group I ribozyme, shows that unfolding of structure can play a large role in RNA folding. Similarly, it appears that Mg2+ slows down the early steps of 30S assembly by preventing unfolding of intermediate structures and possibly by stabilizing misfolded intermediates.