Effects of Novel Polyamines on Cell-Free Polypeptide Synthesis Catalyzed by Thermus thermophilus HB 8 Extract

Abstract

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 quaternary polyamine, tetrakis (3-aminopropyl) ammonium, in thermophile protein biosynthesis. Longer polyamines than triamine supported the polypeptide 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 tetraamine were simultaneously present. The optimum temperature of the reaction supported by the combination of the branched polyamine and spermine was the highest and in accord with the optimum temperature of the bacterial growth. These results suggested an essential role of the quaternary amine in protein synthesis in vivo. This amine effectively stabilized the ternary complex between ribosomes, the messenger, and phenylalanyl-tRNA, and this stabilization may account, at least in part, for its action on the present reaction. In contrast, another branched polyamine, tris (3-aminopropyl) amine supported the activity only moderately even in the presence of another polyamine, though the tris amine stabilized 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 stabilization of the ternary complex. The effects of polyamines on MS2 RNA directed reaction resembled those on poly (U) directed polypeptide synthesis, indicating that polyamines are essential in protein biosynthesis directed by natural messengers in vivo. The quaternary amine inhibited the aminoacylation of tRNAP^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 investigate the effect of polyamines on polypeptide formation, single addition of tetrakis (3-aminopropyl) ammonium was enough for the highest activity, and the synergistic effect disappeared. The results indicate that the role of spermine in the synergism is to relieve the inhibition of aminoacylation caused by the quaternary amine.