Polypeptide-chain elongation promoted by guanyl-5'-yl imidodiphosphate.
In a purified system from Escherichia coli containing ribosomes complexed with poly(uridylic acid) and N-acetyl-phenylalanyl-tRNA, the nonhydrolyzable analog of GTP, guanyl-5'-yl imidodiphosphate (Guo-5'-P2-NH-P), promotes polypeptide synthesis at a rate several times slower than GTP. The activity is completely dependent on elongation factors EF-T (i.e, EF-Ts + EF-Tu) and EF-G. Examination of individual steps of the elongation cycle in partial reactions shows that Guo-5'-P2-NH-P is as efficient as GTP in promoting the EF-T-dependent binding of phenylalanyl-tRNA to the ribosomal A site. In contrast, Guo-5'-P2-NH-P promotes the translocation-dependent binding of phenylalanyl-tRNA to a ribosome complexed with A-site-bound N-acetyl-phenylalanyl-tRNA much more slowly than GTP. This slow rate of binding is due to the presence of EF-G on the ribosome, and not to sluggish translocation, since (a) the rate remains slow even after translocation of N-acetylphenylalanyl-tRNA is completed, (b) it is greatly speeded up by removal of EF-G from the reaction mixture (after translocation has occurred), and (c) it is slowed down again by readdition of the factor. Moreover, with post-translocated ribosomes and in the absence of EF-G, formation of dipeptide subsequent to the EF-T-dependent binding of phenylalanyl-tRNA is much slower when binding of this substrate has been promoted by Guo-5'-P2-NH-P than it is when promoted by GTP. The results suggest that, during polymerization with Guo-5'-P2-NH-P, EF-G and EF-Tu are slowly released from the ribosome and, consequently, the steps of the elongation cycle subsequent to translocation and aminoacyl-tRNA binding (aminoacyl-tRNA binding and peptide bond formation, respectively) are delayed. Thus, durong elongation cycle, GTP hydrolysis is probably essential for fast release of the factors from the ribosome.
D001665:Binding Sites; D004926:Escherichia coli / Q000378:metabolism; D005672:Fusidic Acid / Q000494:pharmacology; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives* / Q000494:pharmacology; D007700:Kinetics; D010441:Peptide Chain Elongation, Translational / Q000187:drug effects*; D010445:Peptide Elongation Factors; D010649:Phenylalanine; D011485:Protein Binding; D012343:RNA, Transfer / Q000378:metabolism; D012270:Ribosomes / Q000187:drug effects / Q000378:metabolism
D010445:Peptide Elongation Factors; D010649:Phenylalanine; D005672:Fusidic Acid; D006160:Guanosine Triphosphate; D012343:RNA, Transfer