RNA codewords and protein synthesis, VII. On the general nature of the RNA code. (1965)

Nucleotide sequences of RNA codons have been investigated recently by directing the binding of C'4-AA-sRNA to ribosomes with trinucleotides of defined base sequence.The template activities of 19 trinucleotidest have been described and nucleotide sequences have been suggested for RNA codons corresponding to 10 amino acids.'-5In this report, the template activities of 26 additional trinucleotides are described and are related to the general nature of the RNA code.Materials and Methods.-Components of reactions: E. coli W3100 ribosomes and sRNA were prepared by modifications of methods described previously.6'Each C14-aminoacyl-sRNA was prepared in the presence of 19 C"2-amino acids.The assay for ribosomal bound C'4-AA-sRNA and components of reaction mixtures have been described.'The characteristics and amounts of labeled AA-sRNA not described' are shown in Table 1.Synthesis and characterization of oligonucleotides: ApG and UpG were obtained from a T-1 ribonuclease digest of RNA, and ApA was prepared by chemical synthesist 10 ApC, ApU, CpA, CpG, GpA, and GpC were obtained from Gallard Schlessinger Corp., but required extensive purification prior to use.GpGpU was obtained by digesting poly UG with pancreatic RNase A; treatment with alkaline phosphatase to remove terminal phosphate groups from the degradation products; and isolation by procedures similar to those described for GpUpU.2The remaining trinucleotides were synthesized frdm the appropriate dinucleoside monophosphate, using either TABLE 1 RADIOACTIVE AMINoAcYLsRNA PREPARATIONS* ApCpA 1 Ap,Cp,A 1.00/0.95/1.05A,pC,pA 1.05/0.90/1.00ApCpC 1 Ap,Cp,C 1.05/1.00/0.95ApC 1.15/2.00ApCpG5 1 Ap,Cp,G 1.00/0.95/1.05A,pC,pG 1.00/0.95/1.00ApCpU 1 Ap,Cp,U 1.00/1.00/0.85ApCpUc 1.00/0.90/1.10ApGpA 1 Ap,Gp,A 1.00/1.00/0.95A,pG,pA 1.00/1.00/1.00ApGpC 1 Ap,Gp,C 1.05/1.00/0.95ApGpCc 1.00/1.00/1.15ApGpUb 1 Ap,Gp,Uc 1.10/1.00/0.95A,pG,pUc 1.00/1.00/1.00ApUpG 1 Ap,UpG 1.00/0.85/1.05A,pU,pGd 1.00/0.90/1.10CpApA 2 Cp,ApA 0.95/1.05/1.00C,pA 1.05/2.00CpApC 2 Cp,Ap,C 1.00/1.10/0.95C,pA,pC 1.00/1.00/1.00CpApGb 2 Cp,Ap,G 1.10/1.00/1.00C,pA,pG 1.00/1.00/1.15CpApU 2 Cp,Ap,U 1.00/1.10/0.90C,pA,pU 1.00/1.05/0.95CpCpA 2e Cp,A 2.00/0.95..... ....... CpGpAb 2 Cp,Gp,A 1.10/1.00/0.95C,pG,pA 1.05/0.95/1.00CpGpC 2 Cp,Gp,C 1.05/1.00/0.80C,pG,pCd 0.90/1.00/1.00CpUpG 2 Cp,Up,G 0.95/1.00/1.10C,pU,pG (.95/1.00/1.1(GpApAl I Gp,Ap,Ad 0.90/1.00/1.10GpAd 1.10/2.00GpApCbo 1 Gp,Ap,C 1.05/1.00/0.90G,pApC 1.05/1.00/0.9(GpApU 1 Gp,Ap,U 1.00/1.00/0.95G,pApUd 1.20/1.00/0.90GpCpU 1 Gp,Cp,U 1.05/1.00/0.90G,pC,pU 0.85/1.00/1.00GpGpUbf 3 Gp,Uf 2.00/0.95G,pG,pUf 0.85/1.00/1.05UpApA 2 Up,Ap,Ad 1.00/1.00/1.10U,pA 0.95/2.00UpApG 2 Up,Ap,G 1.00/0.95/1.00U,pA,pGc 1.00/1.15/0.9(UpCpG 2 Up,Cp,G 1.00/0.90/1.00U,pC,pG 0.95/1.00/1.05UpGpA 2 Up,Gp,A 0.95/1.00/1.10U,pG,pA 0.90/1.00/1.15UpGpCb 2 Up,Gp,C 1.00/0.95/1.05U,pG,pCd 1.10/1.00/0.90a Method 1: primer-requiring polynucleotide phosphorylase.12Method 2: derivative of bovine pancreatic ribonuclease."1Method 3: isolation from a ribonuclease digest of poly-UG.2b Trinucleotide contained a small amount (ca.2%) of an unidentified contaminant.In every case, the chrortiatographic characteristics of the impurity in solvent systems A and B precluded the possibility of it being an oligonucleotide of chain-length greater than two base residues.c The digestion mixture contained another component, 3-5% of the total nucleotide content; the electrophoretic