Untersuchungen zur Biosynthese der Proteine, V. Die Funktion von Nucleinsäuren beim Einbau von Aminosäuren in Proteine in einem zellfreien System ausEscherichia coli

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Abstract
Amino-acids are incorporated into proteins in cell-free extracts from E. coli. The system requires: amino-acids, ATP, amino-acid activating enzymes, soluble RNA, ribosomes and high molecular weight nucleic-acids. Addition of high molecular weight nucleic-acids (messenger RNA and DNA) is essential for incorporation. The following preparations are active: messenger-RNA from E. coli. virus-RNA and double stranded DNA from bacteriophages T2 and T4. Single stranded DNA preparations from bacteriophage [PHI]X-174 or melted and rapidly cooled T2-DNA are inactive. The DNA effect can be observed only during the DNA-dependent synthesis of messenger RNA from 4 ribonucleoside-5''-triphosphates. Synthesis of messenger RNA and amino-acid incorporation are linked in the DNA-dependent system. Ribosomes and messenger RNA act catalytically in the incorporation. Single stranded and double stranded DNA preparations act as primers in biosynthesis of messenger RNA by highly purified DNA-dependent RNA polymerase from E. coli. Synthesis of messenger RNA by RNA polymerase in the presence of double stranded DNA is significantly increased by stoichiometric amounts of spermine. Enzymatic synthesis of RNA is inhibited by actinomycin-C3 in the presence of double or single stranded primer-DNA and by proflavine in the presence of double stranded DNA only. The RNA -product of double stranded DNA is degraded by pancreatic ribonuclease. The RNA-product of single stranded DNA is bound to the primer and thus protected from hydrolysis by ribonucleases. While a high degree of structural integrity is required for activity of DNA in the amino-acid incorporating system, partially hydrolysed DNA still acts as primer for the messenger RNA synthesis by RNA polymerase.