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A cell-free system capable of carrying out amino acid incorporation into peptides was developed from exponentially growing cultures of Pseudomonas aeruginosa . The amino acid incorporation activity required an ATP energy generating system, Mg 2+ , NH 4 + , tRNA and an amino acid mixture for maximum activity. Ribonuclease, puromycin and chloramphenicol inhibited protein synthesis. N -Formylmethionyl-tRNA was synthesized with a P. aeruginosa enzyme system, methionine, P. aeruginosa tRNA, formate and tetrahydrofolic acid. N -formylmethionine was incorporated into peptides by the P. aeruginosa cell-free system. These results indicate that P. aeruginosa extracts contain methionyl-tRNA transformylase and the complete system for the incorporation of N -formylmethionine into peptides.

Transfer of the acetyl- l -leucine (AcLeu) residue from AcLeu-pentanucleotide to synthetic substrates under conditions of the fragment reaction was used to study the specificity of the acceptor site of ribosomal peptidyl transferase. 2′(3′)-O-Aminoacyl nucleosides are the simplest acceptor substrates. Their acceptor activity is dependent on the nature of the nucleoside to which the amino acid residue is bound. The acceptor activity decreased in the sequence 2′(3′)-O- l -phenylalanyladenosine (A-(Phe)) > I-(Phe) > C-(Phe); U-(Phe) was inactive. The presence of a free 2′-hydroxyl group in the ribose moiety of the aminoacyl adenosines was important for the acceptor activity, as shown by the low activity of dA-(Phe) in comparison with A-(Phe). Acceptor activity was influenced by the nature of the side chain of the amino acid residue bound to adenosine: A-(Phe) was a more active acceptor than puromycin, while the acceptor activity of 2′(3′)-O-glycyladenosine A-(Gly) was very low. Free phenylalanine, phenylalanine methyl ester, and adenosine did not act as acceptors. As terminal products of the reactions of AcLeu-pentanucleotide with puromycin, A-(Phe), I-(Phe) and C-(Phe), we isolated AcLeu-puromycin, 2′(3′)-O- acetyl- l -leucyl- l -phenylalanyladenosine (A-(AcLeu-Phe)), I-(AcLeu-Phe) and C-(AcLeu-Phe), respectively.

A rapid assay for tRNA sulfurtransferase from Escherichia coli was developed, reducing the time needed to determine enzyme activity from 11 to 2 h. The reaction measured is the transfer of sulfur from [35S]cysteine to acceptor sites in a thionucleotide-deficient tRNA substrate. Processing is done by binding the product, [35S]-tRNA, to DEAE-cellulose filter disks. The disks are then treated to remove unreacted [35S]cysteine, cysteine-protein adducts and [35S]cysteinyl-tRNA. The DE81 disk assay and the 11-h standard assay are shown to give identical values over a wide range of incubation times and enzyme levels. Incorporation was greater when thionucleotide-deficient tRNA was used as substrate, as compared to fully modified tRNA. [35S]-tRNA was found to be the major reaction product, although some [35S]cysteine was also bound to the filters. The major thionucleoside labeled in nucleoside digests was 4-thiouridine, as determined by Bio-Gel P2 chromatography. We also observed other labeled peaks by this method, in amounts too small for positive identification. This rapid assay should be useful in the purification and study of this uncharacterized class of tRNA modification enzymes.

Amino acids are quantitatively recovered from aminoacyl-tRNA in filter paper strips by treatment with 0.3 m ammonium carbonate. A simple apparatus is described for deaminoacylation and separation of amino acids and tRNA. The amino acids, thus recovered, can be directly analyzed by paper chromatography since ammonium carbonate is removed by lyophilization. Nonvolatile reagents such as Tris-HCl and sodium carbonate also hydrolyze aminoacyl-tRNA, but unless the products of deaminoacylation are desalted, the presence of these reagents gives rise to chromatographic artifacts such as peak broadening, appearance of multiple peaks, displacement of R f values, etc. tRNA remaining after deaminoacylation retains only a small fraction of its original capacity to bind amino acids. Part of this loss of activity appears to be due to structural damage to tRNA in the course of washing and counting procedures and in part, due to the effect of filter paper in shifting the equilibrium of the reaction toward hydrolysis of aminoacyl ester bond.

A semiautomated assay procedure for the determination of aminoacyl-tRNA synthetase activity is described. The assay system employs a Technicon AutoAnalyzer with a continuous filter paper attachment. A larger number of samples can be measured with greater accuracy, reproducibility, and more rapidly than the manual filter paper disk assay system.

A general method for the preparation of tripeptidyl-tRNA is described. The method involves a reaction between N-hydroxysuccinimide ester of N-monomethoxytrityldipeptide and aminoacyl-tRNA. The monomethoxytrityl group is removed from the blocked peptidyl-tRNA under very mild conditions (5% dichloroacetic acid for 5 min at 4°) leaving the tRNA intact. When N-blocked carboxyl-activated tripeptide was reacted with aminoacyl-tRNA and the product formed was treated with dichloroacetic acid, a tetrapeptidyl-tRNA was obtained. The peptidyl-tRNA's described in this communication contained only bifunctional amino acids such as glycine, alanine, valine and phenylalanine. The activated N-blocked dipeptides used were either composed of two identical amino acids as in alanylalanine or of two different amino acids as in glycylphenylalanine.

HeLa cells and L cells were allowed to grow in the presence of l -methioninemethyl- 14 C so that any methylated components of the transfer RNA (tRNA) would be radioactive. The tRNA from these cells was then isolated and a complete analysis was made of the methylated bases present as minor components of the tRNA. For this purpose, the purified RNA was subjected to acid hydrolysis, and the hydrolytic products, including the methylated bases, were partially separated by chromatography on ion-exchange resins. Radioactive (methylated) compounds were then identified by paper Chromatographic analyses and, in some cases, by their chemical characteristics. Methylated bases found in relatively major quantities in both HeLa tRNA and L cell tRNA were: 1-methyladenine, 1-methylguanine, N 2 -methylguan ine, N 2 -dimethylguanine, 7-methylguanine, 5-methylcytosine, and 5-methyluracil (thymine). Methylated bases present in smaller quantities included 1-methylhypoxanthine, 3-methylcytosine, and a base thought to be 3-methyluracil. Evidence was also obtained for the presence in small amounts of 2- O -methylribose residues in both HeLa and L cell tRNA. 5-Hydroxymethyluracil was identified as a minor component of L cell tRNA, but it was not present in HeLa RNA.

1. RNA-iodo-proteinaceous complexes occur in the tRNA fractions isolated from bovine, ovine, and porcine thyroids. The calf thyroid complex has been partially characterized and contains diiodotyrosine and probably the hormone thyroxine in addition to several other amino acids. 2. Different preparative procedures and techniques capable of disrupting a variety of non-covalent bonds and aminoacyl and peptidyl ester bonds had no obvious effect on the calf thyroid RNA-iodo-proteinaceous complex which remained associated with tRNA. 3. Nucleolytic enzymes can degrade the complex to lower molecular weight products that contain iodine, proteinaceous, and RNA components. However, most of the iodine and proteinaceous material is associated with a relatively high molecular weight, ribonuclease-resistant, RNA-containing core. 4. It is concluded that the tRNA fractions of bovine and probably other mammalian thyroid glands contain a low molecular weight RNA linked to proteinaceous material that contains iodinated amino acids.

In vitro methylation of E. coli tRNAfMet fragments or their reconstituted molecule by rat liver methylase showed that (1) the whole tRNA molecule was not necessary for recognition by tRNA methylase, and (2) the extent and the site of methylation of fragments differed depending upon the type of fragment used. This indicates that the conformational structure of the methyl acceptor molecule is important for recognition by the tRNA methylase.