Identification and characterization of an Escherichia coli ribosomal ATPase

grantor: University of TorontoProtein synthesis is a unifying process of all living cells. The macromolecules involved in translation, ribosomes, RNAs and protein factors, as well as the mechanisms of protein synthesis, are all largely conserved across the spectrum of cell types. In this thesis, a ribosome-associated ATPase, RbbA, is identified in 'Escherichia coli' extracts. RbbA can bind both 70S ribosomes and 30S ribosomal subunits. The ATPase activity of RbbA increases two-fold in the presence of purified 70S ribosomes. RbbA cross-reacts with antibody raised against the fungal-specific translation factor EF-3, and like EF-3, RbbA stimulates the synthesis of polyphenylalanine from a poly(U) template 'in vitro'. Cross-linking data show that RbbA lies near 30S ribosomal subunit protein S1 and binds to a poly(G)-rich region of the 30S subunit rRNA, 16S rRNA. This region of the ribosome is known as the decoding region of the ribosome and is important for the binding of aminoacyl-tRNAs to the ribosomal A-site. Upon binding of RbbA, 16S rRNA undergoes a specific conformational change that has been demonstrated previously to affect translational fidelity. EF-Tu, which escorts aminoacyl-tRNAs to the A-site of the ribosome, is also capable of binding with RbbA. Hygromycin, an antibiotic that interferes with protein synthesis by binding to the decoding center of the ribosome, is able specifically to inhibit the ribosomal ATPase activity by displacing RbbA from the ribosome. Together, the data suggest that RbbA, the first ATPase demonstrated to be involved in prokaryotic protein synthesis, plays a major role in protein chain elongation.Ph.D