Function Study On Conserved GTPase EF-G In Protein Synthesis

Ribosome, which is central to protein synthesis, is a big and complicated molecular machine in cells. During translation, the ribosome moves along with mRNA in a one-way direction from5’ to3’and converts the genetic information into a polypeptide chain. A variety of regulated factors function in the process of ribosome translation which is complicated in order to ensure accurate protein synthesis. GTPase is a series of translation factors and plays a crucial role in ptotein synthesis. During the polypeptide chain synthesis, the energy is generated from the process of GTP hydrolysis into GDP and releasing inorganic Pi. There is a GTPase-associated center (GAC) to where many related GTPases bind to regulate the protein synthesis in the ribosome. GAC is responsible for stimulating the hydrolysis activity of GTPase.GTPase is a large group of conservative enzymes in cell which is widely participated and plays an important biological function in cell differentiation, signal transduetion, protein synthesis and transport as well as energy metabolism. GTPase is called molecular switch protein with circulation form function. Elongation factor G (EF-G), elongation factor TU (EF-TU), initiation factor2(IF2) and others are common GTPase which function different in protein synthesis. EF-G is one of the conserved GTPases in protein translation. Sequence and structural data show that it is constituted of five structural domains, in which domains Ⅰ,Ⅱ,Ⅲ. and Ⅴ are homologous to domains Ⅰ to IV of elongation factor4(EF4). EF-G has regions of domains G’ and IV to which EF4does not correspond,while it does not own a similar unique C-terminal domain of EF4. EF-G translocates the mRNA-(tRNA)2complex, moving the peptidyl-tRNA from the A to the P site and the deacylated-tRNA from the P to the E site. In addition, EF-G combines with RRF to participate in ribosome recycling, ensuring the next cycle of protein synthesis.In our study, homology-modelling indicates that the structure of ribosome protection protein C. jejuni Tet(O) has a high similarity to E. coli EF-G and both contain five domains. The two conserved loops of domain Ⅳ in both proteins are different via sequence alignment. So we cloned the mutant gene of EF-G into pET-28a, expressed in E. coli BL21, and then purified proteins. Through C? IP hydrolysis, Poly(U)-dependent poly(Phe) Synthesis. Polysome breakdown assay as well as in vivo experiments, we concluded that EF-G mutants can affect the transloeation of mRNA-(tRNA)2complex, hut have no influence on ribosome recycling.We learned more about the transloeation process through the function study of EF-G chimeras in E. coli protein synthesis in order to lay the theoretical basis lor researches on function mechanism of target antibiotics on prokaryotic ribosomes.