Y. Lapidot

[55] The chemical preparation of acetylaminoacyl-tRNA☆

Publisher Summary This chapter describes the method for the chemical preparation of Acetylaminoacyl-tRNA that is based on the reaction between aminoacyl-tRNA and N-hydroxysuccinimide ester of acetic acid. The active ester is prepared by allowing the acetic acid to react with N-hydroxysuccinimide in the presence of dicyclohexylcarbodiimide. This method was found to be a general one and has been used successfully with aliphatic carboxylic acids—for example, formic, acetic, caprylic, lauric, and palmitic acids as well as with aromatic compounds containing free carboxylic groups. It was found that when N-hydroxysuccinimide ester of [ 3 H]acetic acid was allowed to react with deacylated tRNA, the radioactivity associated with the tRNA was equivalent to one acetyl residue per 100 molecules of tRNA.

Modified aminoacyl-tRNA. II. A general method for the preparation of acylaminoacyl-tRNA.

Abstract A general and relatively simple method for the acylation of aminoacyl-tRNA is described. The synthesis involves a reaction between N- hydroxysuccinimide esters of short- and long-chain fatty acids and aminoacyl-tRNA. The activated fatty acids react specifically with the amino acid attached to the tRNA and do not react with the tRNA proper. The acylated phenylalanyl-tRNA did not lose its biological properties as a result of the acylation reaction. Acylphenylalanyl-tRNA could be bound to ribosomes in a poly (U)-dependent reaction. The tRNA could be recharged with phenylalanine after removal of the acylphenylalanine from the tRNA by mild alkaline hydrolysis.

Antimitogenic effects of interferon and (2′–5′)-oligoadenylate in synchronized 3T3 fibroblasts

Interferons are not only antiviral agents, but are also pleiotropic modifiers of cellular functions [ 1,2]. One aspect of IFN’s cellular activity, is its ability to inhibit normal and tumor cell proliferation [3]. In GO-arrested cells stimulated to grow by different mitogens, hormones or other positive growth-factors, IFN was reported to reduce the rate of cell entry into S-phase [4-71. In some cases, it also extended the S t G2 phases of the cell cycle [8,9]. Several observations have led us to propose that the (2’-S’)-oligoadenylate (oligo(A)) synthetase, which is induced in cells by IFN, is involved in the antimitogenic effects of IFN: First, (2’-5’)oligo(A) produces an antimitogenie effect when applied to intact lymphocytes stimulated by concanavalin A [lo]. Second, growthrelated variations in the synthesis and degradation of (2’~S’)-oligo(A) were demonstrated [ 11,121. Here, we have extended the study of the antimitogenic effect of dephosphorylated (2’-5’)ApApA to serum-stimulated Balb/c 3T3 fibroblasts. Using a series of chemically synthesized derivatives of this oligonucleotide, we show that inhibition of DNA synthesis takes place only if the (2’-5’)-oligo(A) is at least 3 nucleotides long and has a free hydroxyl-group at the 5’-position. We show that the antimitogenic action results from a decrease in the rate of cell entry into S-phase. Finally, we present evidence that the antimitogenic activity of (2’-5’)ApApA is mediated by ribonuclease F (RNase F) activation [ 13,141 and a decrease in protein synthesis during the Cl phase of the cell cycle. 2. Materials and methods

The synthesis of glycyl-l-phenylalanyl-sRNA

Abstract Glycyl- l -phenylalanyl-sRNA was prepared by acylation of l -phenylalanyl-sRNA. The method involves the use of N-hydroxysuccinimide ester of N-protected glycine as a highly specific reagent which acylates only the amino group of the phenylalanine bound to the sRNA and does not react with any of the functional groups of sRNA. The amino group of glycine was protected with mono-p-methoxytriphenylmethyl. This protecting group was found to be stable during the acylation reaction and could be removed from the dipeptidyl-sRNA under very mild conditions (5 % trichloroacetic acid for 5 min at 4°), leaving the sRNA intact.

Peptidyl-tRNA. VII. Substrate specificity of peptidyl-tRNA hydrolase.

Abstract The enzymatic hydrolysis of peptidyl-tRNA is described. The hydrolysis rates of peptidyl-tRNA with different peptide chain lengths containing free and blocked α-amino groups are compared to the hydrolysis rates of N-acylaminoacyl-tRNA. It is shown that the hydrolysis rate of peptidyl-tRNA containing two peptide bonds is considerably higher than that of N-acylaminoacyl-tRNA. Moreover, the hydrolysis rate of different peptidyl-tRNAs depends on the peptide chain length. Thus, Gly2-Phe-tRNA is hydrolyzed faster than GlyPhe-tRNA, and Gly4Phe-tRNA is hydrolyzed faster than Gly2Phe-tRNA. The apparent Km and vmax values for Ac-Leu-tRNA are compared to those of Ala2Leu-tRNA.

Modified aminoacyl-tRNA: III. A general procedure for the synthesis of dipeptidyl transfer RNA☆

Abstract A general method for the preparation of dipeptidyl transport RNA (tRNA) is described. The synthesis involves a reaction between N-hydroxysuccinimide esters of N-monomethoxytritylamino acids and aminoacyl-tRNA. The activated N-protected amino acids react specifically with the amino group of the amino acid attached to the tRNA and do not react with any of the functional groups of the tRNA. [14C]Valyl-, [14C]phenylalanyl- and [14C]methionyl-tRNA could be 100 % transformed into the corresponding dipeptidyl-tRNA when they were reacted with activated, N-protected glycine and alanine. With the activated N-protected phenylalanine only 80–85 % of dipeptidyl-tRNA was formed. Mono-p-methoxytrityl, notwithstanding its bulkiness12, was found suitable for protecting the amino group of several amino acids. In all the reactions tested, the monomethoxytrityl remained attached to the amino group during the acylation and could be removed from the dipeptidyl-tRNA under mild acidic conditions. The tRNA of the dipeptidyl-tRNA did not lose its biological properties as a result of the chemical synthesis. Dipeptidyl-tRNA could be bound to ribosomes in a polyuridylic acid dependent reaction, and the tRNA could also be recharged with amino acid after removal of the dipeptide from the tRNA by mild alkaline hydrolysis.

The chemical synthesis and the biochemical properties of peptidyl-tRNA.

Publisher Summary This chapter explores that the process of protein biosynthesis, the study of the chemical, physical, and biochemical properties of peptidyl-tRNA is of considerable interest. A limited number of peptidyl-tRNAs have been isolated from in vitro ribosomal systems, using defined messenger RNA or peptide-synthesizing systems synchronized by limiting one of the components necessary for continuous synthesis. A more general approach for the preparation of peptidyl-tRNAs is based on the condensation of an N-blocked carboxyl-activated amino acid with aminoacyl-tRNA arid subsequent removal of the N-blocking group. Two major problems are involved in the chemical preparation of peptidyl-tRNA. First, the N-blocked carboxyl-activated amino acid should react specifically with the amino group of the amino acid attached to the tRNA and with no other functional group of the tRNA molecule. Second, the acylation reaction and the removal of the N-blocking group from the N-blocked peptidyl-tRNA should proceed under mild conditions, leaving the tRNA undamaged. An enzymatic activity that hydrolyzes the ester bond between the peptide and the tRNA has been found in several biological systems. A fraction containing this activity was extensively purified from E. coli and its substrate specificity has also been discussed. The chapter concludes that detailed physical studies are necessary to solve this problem.

Peptidyl transfer RNA VI. The chemical synthesis of tri- and tetrapeptidyl transfer RNA

Abstract 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-tRNAs 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.

Correlation of the differences in conformation between 2′–5′ and 3′–5′ dinucleoside monophosphates with their behaviour on a Sephadex LH-20 column

Abstract The separation of 2′–5′ dinucleoside monophosphates from their 3′–5′ isomers on Sephadex LH-20 column is described. In addition, the CD spectra of all the different dinucleoside monophosphates are given. The correlation of the differences in conformation between the 2′–5′ and the 3′–5′ dinucleoside monophosphates with their behaviour on the Sephadex LH-20 column is discussed. It is shown that nucleotides do not behave on the Sephadex LH-20 column according to the rules of gel filtration and it is suggested that the separation of the different dinucleoside monophosphates is dictated by the conformation, which affects the adsorption of the nucleotidic material to the column.

Peptidyl-tRNA: XI. The chemical synthesis of phenylalanine-containing oligopeptidyl-tRNA

Abstract The synthesis of phenylalanine-containing peptidyl-tRNA is described. The method involves a reaction between the N-hydroxysuccinimide ester of N-o- nitrophenylsulfenyl- l -Phe n (n = 1–2) or N-o- nitrophenylsulfenyl- l -Phe n -Gly (n = 1–3) and aminoacyl-tRNA and subsequent removal of the N-protecting group by treatment with Na2S2O3 in the presence of 6 M urea. Phe-Phe-[14C]Phe-tRNA was also prepared by the reaction between the N-hydroxysuccinimide ester of N-monomethoxytrityl-Phe-Phe and [14C]Phe-tRNA. The monomethoxytrityl group was removed from the blocked peptidyl-tRNA under mild acidic conditions (5% dichloroacetic acid at 4°).