Structural And Functional Studies Of A Ribosome-associated GTPase EngA In Escherichia Coli

The ribosome is a large molecular machine responsible for biological protein synthesis in all living cells. The assembly of ribosome is a complex process, which requires a large number of functional diverse factors, including many ATP-dependent RNA helicases and GTPases. Many ribosome associal GTPase, with proposed functions in ribosome biogenesis, are believed to be key players that couple between ribome assembly process and various growth control pathways. EngA is an essential GTPase in bacteria. It contains two tandem GTP binding domains, and being one-of-a-kind in all the known GTPases. EngA is essential for the ribosomal large subunit maturation. However, little is known how EngA functions at molecular lever.In this thesis, biochemical studies and electron microscopy and single particle analysis method are used to study the structures of 50 S ? EngA complexes and interaction between them. We determined two cryo-EM structures of EngA ? 50 ?GMPPNP complex and EngA?50S?GDP complex. The two stuctures show that EngA binds to the 50 S in two separate sites in different nucleotides conditions. In the presence of GMPPNP, EngA binds to the peptidyl transfersae center and induces dramatic conformational changes on the 23 S rRNA, virtually returning the 50 S subunit to a state similar to that of the late-stage 50 S assembly intermediateds. The two GTP binding domains exhibit a pseudo-two-fold symmetry in this specific conformation. In the presence of GDP, EngA binds beside the the peptide tunnel exit, displaying apparent incompatibility with many nascent polypeptide interacting factors. In this thesis, interaction between EngA and mature 50 S was studied by biochemical methods. The study shows in the presence of the 50 S subunit, the GTPase activity of EngA is enhanced. it indicates that the 50 S subunit might reorganize the two GTPase domains into a pseudo-dimeric conformation such that they might be mutually activated in a trans manner. Interaction between EngA and two immature 50 S was studied, too. Result of the pellet assay shows EngA had increased affinity with immature 50 S particles from an rrmJ knockout strain. A certain conformational immature large ribosomal subunit is the native substrate for EngA.These structural and biochemical information gave important clues on how the 50 S subunit interact with EngA. Our results provide important insight into the molecular role of EngA.