H.G. Khorana

Studies on polynucleotides

A ribopolynucleotide containing the repeating trinucleotide sequence, adenylyl-adenylyl-guanylate (poly AAG) has been shown to direct the synthesis of polyly-sine, polyarginine and polyglutamate in the in vitro amino acid incorporation system. In experiments on the stimulation of the binding of [ 14 C]aminoacyl-sRNA to ribosomes the following results were obtained. (1) Poly AAG stimulated the binding of lysyl-, glutamyl-, arginyl- and aspartyl-sRNA. (2) The trinucleotide, GpApA, was specific in inducing the binding of glutamyl-sRNA and the trinucleotide, ApApG, was specific in inducing.the binding of lysyl-sRNA to ribosomes. The new codon sequences ApApG for lysine and GpApA for glutamic acid are therefore proposed. (3) The codon sequence ApGpA is proposed for arginine, despite the failure of this trinucleotide to stimulate the binding of arginyl-sRNA to ribosomes. (4) GpApA also stimulated the binding of aspartyl-sRNA to ribosomes. In the in vitro amino acid incorporation experiments, however, no polyaspartate synthesis has so far been detected in the presence of poly AAG.

Studies on polynucleotides: XCVI. Repair replication of short synthetic DNA's as catalyzed by DNA polymerases☆☆☆

Abstract Repair replication of short synthetic DNAs corresponding to parts of the gene for the major yeast alanine transfer RNA has been studied. The enzymes used were the Escherichia coli, Microccua luteus and T4 DNA polymerases, similar results being obtained with all the three enzymes. The DNAs used (Fig. 1) were: four double-stranded DNAs with the termini containing the 5′-hydroxyl group protruding by one, three, four or ten nucleotide units and two single-stranded DNAs 29 units long which are capable of folding back on themselves. The aspects inve tigated were: (1) completion of repair, (2) the minimum size of the polynucleotide chains required as primers and those which can serve as templates and (3) the minimum size of the primers which can abolish hairpin formation so as to give the “normal” repair replication. Repair replication of DNAs (DNA-I to DNA-IV: Fig. 1) was characterized to be essentially complete. The minimum size of the primer for repair replication of an extended single-stranded deoxypolynucleotide was ascertained to be about five to seven units long, while the primers required to overcome the hairpin formation in DNA-V and DNA-VI were found to be about twelve units long. Thus, in the presence of a suitable primer, the nucleotide incorporation which occurs in DNA-V in the absence of added primer was completely replaced by incorporation expected for normal repair replication. The minimum size of a chain which can serve as a template was also found in the present work to be about 12 units long.

Studies on polynucleotides: LXVII. Initiation of protein synthesis in vitro as studied by using ribopolynucleotides with repeating nucleotide sequences as messengers☆☆☆

Abstract All of the 64 ribotrinucleotides were tested for the binding of fmet-tRNAF to ribosomes. The trinucleotides AUG, GUG, UUG, ACG, CUG, GCG, UGG, UCG and AGG stimulated the binding, the first three giving the maximum effect. The non-formylatable species (met-tRNAM) was recognized by the triplets AUG and GUG. Polypeptide synthesis in Escherichia coli cell-free system at 0.004 m -Mg2+ concentration proceeded only when messengers contained initiator codons; it also required the presence of fmet-tRNAF and additional stimulation was obtained by the addition of a protein fraction isolated from ribosomal washings. Poly r-UG containing repeating dinucleotide sequence directed the synthesis of fmet-(Cys-Val-)n, and the N-terminal sequence was established as fmet-Cys-Val-Poly r-AUG directed polymethionine synthesis required fmet-tRNAF in addition to met-tRNAM needed for the incorporation of internal methionine. At low Mg2+ ion concentration poly r-GUA specifically stimulated the incorporation of valine, dependent on fmet-tRNAF. Although the triplet UUG stimulated the binding of fmet-tRNAF to ribosomes, poly r-UUG failed to stimulate fmet incorporation and no fmet-tRNAF-dependent polypeptide synthesis at 0.004 m -Mg2+ concentration was observed. From these results the triplets AUG, GUG and GUA are concluded to be codons for the initiation of polypeptide chains by fmet-tRNAF.

Studies on polynucleotides: XLVIII. The in vitro synthesis of a co-polypeptide containing two amino acids in alternating sequence dependent upon a DNA-like polymer containing two nucleotides in alternating sequence

The DNA-like polymers, poly d-TC:AG and poly d-TG:AC, which contain in each strand only two bases in alternating sequence, have been used as templates for DNA-dependent RNA polymerase. In this way, four single-stranded long-chain ribopolynucleotides, poly UC, poly AG, poly UG and poly AC containing in every case the two nucleotides in alternating sequence have been prepared. The messenger activity of poly UC in the Escherichia coli cell-free amino acid incorporation system has been studied. The ribopolynucleotide stimulates the incorporation of only two amino acids, serine and leucine, in equimolar amounts. The product of the reaction has been characterized as a co-polypeptide containing serine and leucine in alternating sequence.

Specificity of sRNA for recognition of codons as studied by the ribosomal binding technique

Multiple sRNA species which are specific for individual amino acids have been purified from yeast and Escherichia coli sRNA using techniques of column chromatography or countercurrent distribution. The purified sRNA fractions, after charging with radioactive amino acid, have been tested for binding to ribosomes in the presence of the trinucleotides assigned as codons for the respective amino acids. From the stimulation of binding, it is concluded that (1) an sRNA species shows strict specificity for the recognition of the first letter of a codon; and (2) an sRNA species often can recognize multiple codons differing in the third letter. The total results, which are summarized in Table 4, appear to provide support for the postulates of the wobble hypothesis ( Crick, 1966 ).

Studies on polynucleotides: L. Synthetic deoxyribopolynucleotides as templates for the DNA polymerase of Escherichia coli: A new double-stranded DNA-like polymer containing repeating dinucleotide sequences

Chemically synthesized short-chain deoxyribopolynucleotides d(TG) 2–6 , containing alternating thymidylate and deoxyguanylate units, and d(AC) 2–6 , containing alternating deoxycytidylate and deoxyguanylate units, have been studied as templates for the DNA polymerase of Escherichia coli . In the presence of members of both series of polynucleotides, the enzyme catalyzes extensive polymerization of deoxyribonucleoside triphosphates of the four bases, T, C, A and G, to give a high molecular DNA-like polymer (poly d-TG : AC). The polymer possesses a highly ordered double-stranded structure, with one strand containing strictly alternating thymidylate and deoxyguanylate units and the complementary strand containing alternating deoxycytidylate and deoxyadenylate units. Poly d-TG : AC can be re-utilized for more synthesis in the presence of the four deoxyribonucleoside triphosphates. Characteristics of the polymer, which are described, include the following. (1) The temperature-absorbance profile shows a sharp transition which is fully reversible; (2) the two complementary strands can be separated by centrifugation in an alkaline cesium chloride density-gradient to yield well-defined bands; (3) electron microscopy shows poly d-TG : AC to consist of molecules with average chain length of 0·5 μ , some of which are branched; (4) the molecular weight of the polymer is at least one million.

Studies on polynucleotides: LXX. Synthetic deoxyribopolynucleotides as templates for the DNA polymerase of Escherichia coli: DNA-like polymers containing repeating tetranucleotide sequences☆☆☆

Abstract Short-chain deoxyribopolynucleotides containing repeating trinucleotide sequences serve as templates for the DNA-polymerase-catalyzed polymerization of deoxyribonucleoside triphosphates. The products are double-stranded DNA-like polymers containing, in both of the complementary strands, repeating trinucleotide sequences which were present in the short-chain templates. Four DNA-like polymers were thus prepared: poly dTTC:dGAA, poly dTTG:dCAA, poly dTAC:dGTA and poly dATC:dGAT. In every case, it was necessary to use as the templates, short segments of chain length 9 to 15 nucleotide units corresponding to both of the complementary strands of the desired DNA-like polymer. All of the polymers were shown to contain repeating trinucleotide sequences by comprehensive nearest-neighbor frequency analyses. Three of the four DNA-like polymers effectively served as templates for the synthesis of more of the same polymer in the presence of DNA-polymerase. Characterization of the DNA-like polymers with respect to molecular weight and buoyant density in a cesium chloride density-gradient is described in an accompanying paper.

Studies on polynucleotides: LXXIV. Direct translation in vitro of single-stranded DNA-like polymers with repeating nucleotide sequences in the presence of neomycin B

Abstract The messenger activity of single-stranded deoxyribopolynucleotides containing repeating nucleotide sequences, poly d-A, poly d-T, poly d-CA and poly d-TG, has been examined in the presence of the antibiotic neomycin B in the cell-free protein-synthesizing system from Escherichia coli B. Except for poly d-A, which failed to stimulate the incorporation of any amino acid, excellent incorporations were observed with the other deoxyribopolynucleotides. The amino acids incorporated were the same as expected for the corresponding ribopolynueleotides but in the absence of neomycin B. However, cases of occasional specific misreading, in general at very low efficiency relative to normal incorporations, were observed. Thus, poly d-CA stimulated the incorporation of only two amino acids, threonine and histidine. The product of the reaction was characterized as a copolypeptide containing threonine and histidine in alternating sequence. Poly d-TG stimulated the incorporation of the expected amino acids, cysteine and valine, that of alanine and arginine to a lesser extent and those of glutamic acid and glycine to a slight extent. Poly d-T stimulated mostly the incorporation of phenylalanine but slightly that of leucine. A number of related antibiotics, excepting gentamicin, failed to stimulate phenylalanine incorporation with poly d-T. Except for poly d-A, the deoxyribopolynucleotides stimulated the binding of the appropriate aminoacyl-tRNAs to ribosomes, and neomycin B was not required for this step. The initiator tRNA, formylmethionyl-tRNA, was also bound to ribosomes by poly d-TG: polypeptide synthesis directed by this deoxypolynucleotide in the presence of neomycin B at low magnesium ion concentration was dependent on the presence of the initiator-tRNA.

Studies on polynucleotides. LVI. Further syntheses, in vitro of copolypeptides containing two amino acids in alternating sequence dependent upon DNA-like polymers containing two nucleotides in alternating sequence.

The double-stranded DNA-like polymers, poly d-TC : AG and poly d-TG : AC, which contain, in each strand, the two specified deoxyribonucleotides in alternating sequence, have previously been shown to serve as templates for DNA-dependent RNA polymerase; and the products formed in the presence of sets of two appropriate ribonucleoside 5′-triphosphates, have been characterized as poly UC, poly AG, poly UG and poly AC containing, in every case, the two ribonucleotides in alternating sequence ( Nishimura, Jones & Khorana, 1965 ). The messenger activity of poly UG, poly AC and poly AG in the Escherichia coli cell-free amino acid-incorporating system has now been studied. Poly UG stimulates the incorporation of valine and cysteine, poly AC stimulates the incorporation of threonine and histidine and poly AG stimulates the incorporation of glutamic acid and arginine. The amino acid-incorporations have the following common features: (1) only two amino acids are incorporated; (2) the incorporation of the two amino acids is largely interdependent; and (3) the incorporation of the two amino acids occurs in equimolar amounts. The products of the in vitro syntheses have all been characterized, by chemical and/or enzymic methods, as copolypeptides containing the two amino acids in alternating sequence. From a combination of these and previous results, the following codon assignments can be made unequivocally: UGU, cysteine; GUG, valine; CAC, histidine; ACA, threonine; AGA, arginine and GAG, glutamic acid.

Studies on polynucleotides: LXXIII. Synthesis in vitro of polypeptides containing repeating tetrapeptide sequences dependent upon DNA-like polymers containing repeating tetranucleotide sequences: Direction of reading of messenger RNA☆☆☆

The double-stranded DNA-like polymers, poly d-TATC:GATA‡ and poly d-TTAC:GTAA, which contain in each strand repeating tetranucleotide sequences indicated by the nucleotide initials, have been used as templates for DNA-dependent RNA polymerase. By providing in the reaction mixture the three ribonucleoside triphosphates necessary for the transcription of only one of the two strands, the following four single-stranded ribopolynucleotides have been prepared: poly r-UAUC, poly r-GAUA, poly r-UUAC and poly r-GUAA. All of the ribopolynucleotides were shown to contain repeating tetranucleotide sequences by nearest-neighbor frequency analysis. In the cell-free amino acid-incorporating system from Escherichia coli B, poly r-UAUC and poly r-UUAC, each directed the incorporation of only four and three amino acids respectively. As expected from the three-letter, non-overlapping code, the polypeptidic product formed by poly r-UAUC was shown to have the repeating tetrapeptide sequence, Tyr-Leu-Ser-Ile, whereas the polypeptidic product from poly r-UUAC had the repeating tetrapeptide sequence, Leu-Leu-Thr-Tyr. With poly r-GUAA and r-GAUA as messengers, no acid-insoluble polypeptides were formed. The present results provide (1) further independent proof of the direction of reading of the messenger RNA and (2) confirmation of a total of ten codon assignments, including the nonsense triplets UAA and UAG, in the strain B of E. coli