Tokumasa Nakamoto

Abstract Escherichia coli ribosomes were made deficient in a 50 S ribosomal protein component by treatment with ethanol and NH4Cl and their ability to carry out a number of partial reactions involved in polypeptide synthesis was examined. The ribosomes were found to be competent in all reactions in which the ribosome alone participated, but were markedly deficient in all reactions involving an interaction of the ribosome, GTP, and either of the supernatant factors T and G. Nonenzymatic phenylalanyl-tRNA and N-acetylphenylalanyl-tRNA binding and the peptidyl transferase activity were essentially intact in these ribosomes; enzymatic binding of phenylalanyl-tRNA and translocation occur, but at markedly reduced rates; uncoupled GTPase activity associated with G factor and the ability to bind [3H]GTP in the presence of G factor and fusidic acid were virtually abolished; and uncoupled GTPase activity associated with T factor was diminished to about 15% of the activity present with unextracted ribosomes. Polyphenylalanine synthesis and all the partial reactions were completely or substantially restored by readdition of the ribosomal extract.

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The requirement for a deoxypolynucleotide primer raised the question of the role played by this polymer in RNA synthesis. A recent report from this laboratory demonstrated that the base composition of RNA, prepared with a partially purified extract from Micrococcus lysodeikticus, was similar to and dependent upon the composition of the primer.8 Similar results have been obtained by Stevens9 and Furth et al. 1, 11 with extracts prepared from Escherichia coli. Cellular RNA similar in base composition to DNA has been identified in various organisms by a number of investigators.2,' 1 Recently, Hall and Spiegelman have shown that the RNA which appears after T2 phage infection of E. coli forms complexes with denatured phage DNA.14 Such complexes are believed to occur only between strands of complementary base sequences and point out the complementary relationship between the deoxyand ribopolynucleotides. The existence of similar DNA and RNA molecules in nature has added impetus to the idea that genetic information is transmitted to other cellular sites by so-called messenger-RNA.' 16 The discovery of a DNA-dependent RNA polymerase has stimulated speculation that this enzyme may be responsible for the synthesis of this specific-RNA. So far, no direct experimental proof has been offered to support these speculations. Weiss and Nakamoto have presented preliminary evidence indicating that the relative position of ribonucleotides in enzymatically prepared RNA is determined by the primer used for the reaction.8 This information, along with the similar base composition found, suggests, but does not prove, that synthesis occurs with replication of the nucleotide sequences of the primer. Absolute and unequivocal proof of replication can only be supplied by determining the sequential arrangement of bases in both primer and RNA product. In the absence of methods adequate to perform such analysis, a complete nearest-neighbor frequency study for the 16 possible nucleotide pairs in RNA has been determined for RNA's prepared with