Study Of The Large Ribosomal Subunit Assembly In Bacillus Subtilis

Ribosome is a cell organelle responsible for protein synthesis, made up of a large and a small subunit. Every subunit is a ribonucleoprotein complexes, consisting of one or two long strand rRNA and a series of ribosomal proteins. Ribosome assembly is a process fundamental for all celluar activities. The efficiency and accurancy of the process is highly regulated. Ribosome assembly consist of various assembly events, including rRNA folding and binding of ribosomal proteins. In vivo, there are a lot of assembly factors involved in this process. Deletion or depletion of these assembly factors will seriously affect the maturation of the ribosome, result in accumulation of assembly intermediates.As a GTPase, YlqF is an assembly factor, participating in large ribosomal subunit in Bacillus subtilis. Depletion of YlqF induces accumulation of premature 50 S subunit, 45 S. In this paper, we combined quantitative mass spectrometry and three-dimensional cryo-electron micrography(Cryo-EM) to identify composition of ribosomal proteins and conformation 23 S rRNA in 45 S particles. Our quantitative mass spectrometry data indicate that L28, L16, L33, L36 and L35 are serious reduced in the 45 S particles. This protein spectrum is similar to many other 50 S precursors, indicating the presence of a conserved rate-limiting step in the late-stage assembly of large subunit. The structural analysis indicates that 45 S particles consisit of two conformations of assembly intermediates, State I and State II. Both conformations seriously lack some ribosomal proteins, consistant with quantitative mass spectrometry data. The functional domains are not well assembled in both conformations, such as PTC or subunit interface, demonstrating that they are both defective in both subunit association and tRNA binding. The two conformations differ in the maturation state of CP and H38. For state II, CP is highly flexible and H38 rotates toward the L7/L12 direction; Then for state I, CP and H38 have been mutaual stabilized, and H38 is in a native-like state. In combination of the analysis above, we provide a model for the late-stage assembly of large subunit, which be made up of two consecutive events, transition from State II to State I and from State I to mature 50 S. The transition from State II to State I corresponds to the well assembly of 23 S rRNA, in which the redirection of H38 is likely the major rate-limiting step. transition from State I to mature 50 S corresponds to the binding of ribosomal proteins.In addtion, in this paper, we also performed structural study on the interaction between YlqF and 50 S or 45 S particles. We applied Cryo-EM to analyze the structures of YlqF-50 S or YlqF-45 S complexes assembled in vivo or in vitro. Cryo-EM data indicate that the binding site of YlqF are all on 23 S rRNA, especially on H38/CP domain, which suggest that YlqF is likely a rRNA chaperone, faciliting the corrected assembly between H38 and CP.