EFFECTS OF NOVEL POLYAMINES ON CELL-FREE POLYPEPTIDE-SYNTHESIS CATALYZED BY THERMUS-THERMOPHILUS HB8 EXTRACT

Effects of novel, naturally occurring polyamines on protein synthesis catalyzed by Thermus thermophilus cell-free extract were investigated. The results revealed the physiological importance of a branched quate rnary polyamine, tetrakis(3-aminopropyl) ammonium, in thermophile prot ein biosynthesis. Longer polyamines than triamine supported the polype ptide synthesis at high temperature, though both the activity and the optimum temperature varied depending on polyamines added. The highest activity was found when tetrakis(3-aminopropyl)ammonium and a tetraami ne were simultaneously present. The optimum temperature of the reactio n supported by the combination of the branched polyamine and spermine was the highest and in accord with the optimum temperature of the bact erial growth. These results suggested an essential role of the quatern ary amine in protein synthesis in vivo. This amine effectively stabili zed the ternary complex between ribosomes, the messenger, and phenylal anyl-tRNA, and this stabilization may account, at least in part, for i ts action on the present reaction. In contrast, another branched polya mine, tris(3-aminopropyl)amine supported the activity only moderately even in the presence of another polyamine, though the tris amine stabi lized the ternary complex as effectively as the quaternary amine. This result suggests the presence of another essential site for polyamine action in the thermophile polypeptide synthesis, in addition to the st abilization of the ternary complex. The effects of polyamines on MS2 R NA directed reaction resembled those on poly(U) directed polypeptide s ynthesis, indicating that polyamines are essential in protein biosynth esis directed by natural messengers in vivo. The quaternary amine inhi bited the aminoacylation of tRNA(Phe), and the inhibition was canceled by the addition of another polyamine. When phenylalanyl-tRNA instead of free phenylalanine was added to the reaction mixture in order to in vestigate the effect of polyamines on polypeptide formation, single ad dition of tetrakis(3-aminopropyl)ammonium was enough for the highest a ctivity, and the synergistic effect disappeared. The results indicate that the role of spermine in the synergism is to relieve the inhibitio n of aminoacylation caused by the quaternary amine.