Interactions of guanosine triphosphate analogues with elongation factor G of Escherichia coli.
Previous studies from this laboratory have shown that the GTP analogue guanosine 3'-diphosphate 5'-triphosphate (pppGpp) was almost without activity in the translocation reaction catalyzed by elongation factor G (EF-G), while it was fully active with elongation factor T (EF-T) and initiation factor 2 (IF-2). To assess the importance of the 3'-ribose hydroxyl itself in the translocation reaction, we examined polyphenylalamine synthesis and EF-G-dependent formation of N-acetylphenylalanylphenylalanylpuromycin (Ac-Phe2-puromycin) supported by 3'-deoxyguanosine 5'-triphosphate (3'dGTP) and 3'-deoxy-3'-aminoguanosine 5'-triphosphate (3'dNH2GTP). Like pppGpp, these nucleotides were similar to GTP in EF-T and IF-2dependent reactions. We also examined the ability of the dialcohol derived from GTP by periodate oxidation and borohydride reduction (rroGTP) and of ITP to support translocation. These compounds had shown significant, although reduced, activity with EF-T and IF-2. A spectrum of activity was found with these compounds in both poly(Phe) synthesis and Ac-Phe2-puromycin formation. All had significantly reduced activity relative to GTP, but all were significantly more active than pppGpp. A surprising finding was that the activities of all analogues relative to GTP were dependent on the reaction temperature in both poly(Phe) synthesis and Ac-Phe2-puromycin formation; these relative activities were significantly lower at 8 degrees C after than 37 degrees C. Furthermore, the extent of poly(Phe) synthesis with the analogues relative to GTP could be significantly affected by the amounts of EF-G and EF-T in the reaction mixture. Differences were minimized in the presence of rate-limiting EF-T and saturating EF-G and maximized in the presence of rate-limiting EF-G and saturating EF-T. This effect of reducing the level of EF-G in the reaction mixture thus mimicked the effect of lowering the incubation temperature, and a similar observation was made for Ac-Phe2-puromycin formation. GTP-supported formation of Ac-Phe2-puromycin at 8 degress C could be inhibited by 3'dNH2GTP, 3'dGTP, pppGpp, and ITP: these compounds were better inhibitors than they were substrates. Inhibition by other nucleotides was also noted. The GTP analogues were compared to GTP as substrates in EF-G-dependent reactions uncoupled from protein synthesis. Fusidic-acid-dependent binding of nucleotides to ribosomes and ribosome-dependent catalytic nucleotide hydrolysis were both examined, and the activity of the nucleotides as substrates in these ractions showed little correlation with their ability to support translocation.