Polypeptide‐Chain Elongation Promoted by Guanyl‐5′‐yl Imidodiphosphate

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Abstract
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, during the elongation cycle, GTP hydrolysis is probably essential for fast release of the factors from the ribosome.