Study On Structures And Functions Of Ribosome Translation Factor EF-G And EF4

In all the kingdoms,ribosomes are protein synthesis machines andtranslate the genetic code in mRNA into polypeptide.Protein translation is a complicated process with high speed andfidelity and lots of translation factors are involved in.EF-G is the only factor involved in two phases: in elongation phase,EF-G helps the(tRNA)2·mRNA duplex to move forward by one codon;in ribosome recycling phase,EF-G and RRF dissociate the post termination complex(PoTC)into ribosomal subunits,and release mRNA and deacylated-tRNA,facilitating the ribosome for the next-round translation.EF4 exists widely in bacterial translation system and specifically catalyzes the back-translocation of tRNAs.In this research,we investigate the molecular roles of EF-G and EF4 in elongation and ribosome recycling phases by combining cryo-EM single particle three-dimensional reconstruction,biochemistry and molecular biology methods.The results show that loop II deletion or S588 P mutation of EF-G greatly destroys itspolysome breakdown ability and translocase activity.One novel PRE·EF-G complex was characterized which may reflect an early state after initial binding of EF-G.In the structure of PoTC·EF-G(S588P)·GDPNP complex,loop II(S588P)of EF-G neighbors to the groove of H69/h44 close to B2 a bridge.Therefore,we suppose that EF-G is involved in the final subunits dissociation process in addition to facilitating the orientation of RRF.During the process,loop II plays a mechanistic role to disrupt the interactions in the vicinity of decoding center(DC),in a similar manner as its role in translocation.Both in translocation and ribosome recycling,interactions between loop II and DC constitute a threshold event,which reflects a common aspect of EF-G function in the two phases.In the EF4 part,we resolved two distinct EF4-induced translating-ribosome intermediates.In Post-EF4,the interaction between the CCA-end of peptidyl-tRNA in P-site and P loop in PTC is disrupted.In Pre-EF4,peptidyl-tRNA was stabilized in the A/4-site and the tRNA acceptor arm interacts with C-terminal domain(CTD)of EF4 in multiple sites.In contrast to the EF-G catalyzed forward translocation of tRNA's anticodon arm,EF4 disengages the acceptor arm on ribosome50 S to induce the back-translocation of tRNA.Our findings provide a better understanding of protein translation.