Jiří Smrt

The binding site for the 3′-terminus of aminoacyl-tRNA in the molecule of elongation factor Tu from Escherichia coli

The mRNA-directed binding of aminoacyl-tRNA to the ribosome requires a special protein factor EF-T, and GTP. A ternary complex between aminoacyl-tRNA, EF-T, and GTP is formed as an intermediate from which the ~~oacyl-t~A is transfe~ed to the ribosomal recognition site (reviewed [ 11). it is the main feature of EF-T, that in the form of EF-T, .GTP it is able to discriminate between aminoacylated and nonor acylaminoacyfated tRNAs [2-4]. This provided a basis for the accumulat~g evidence that the 3’-terminus of aminoacyl-tRNA is involved in the recognition reaction between aminoacyl-tRNA and the elongation factor T, [S-7]. Therefore, we prepared two analogs of the 3’oterminus of phenylalanyl-tRNA, 2’(3’)0L-phenylalanyladenosine and cytidylyl-(3’ + 5’~2’(3’)U-L-p~enylalanyladenosine and tested them for the ability to replace aminoacyl-tRNA in protecting the aminoacyltRNA binding site of EF-T, from inactivation by N-tosyl-L-phenylalanylchloromethane. The results presented here show that: (1) A-Phe and CpA-Phe can interact with the aminoacyl-tRNA binding site of EF-T,; (2) The SH group in the aminoacyl-tRNA binding

The reaction of 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane with some open chain polyhydroxy compounds

Abstract 1,3-Dichloro-1,1,3,3-tetraisopropyldisiloxane (I) forms an 8-membered ring (IIIa) with glycerol, a 7-membered ring (Va) with D-erythropentose phenylosazone (IV) and linear polymers with 1,4-butanediol resp.

Inhibition of protein synthesis by exogenous A2'p5'A2'p5'A (2-5 A core) and its bis-phosphoramidate analog in intact mouse lymphocytes, hepatocytes and bone marrow cells

Interferon-treated cells are capable of synthesizing at least 3 enzymes which are involved in normal cell proliferation and anti-viral immunity [l-4]. One of these enzymes, interferonand dsRNA-dependent oligoisoadenylate synthetase is responsible for a polymerization of ATP resulting in a series of (2’ + 5’)-linked oligoadenylic acid 5’-triphosphates. The major active species is the trimer, pppA2’p5’A2’p5’A(2-5 A), which is able to inhibit protein synthesis in cell-free system at subnanomolarievels [5-7 1. This inhibition is mediated by activation of a riboendonuclease which degrades cellular mRNA [g-lo]. In [l l] it was shown that (2’ + 5’)-oligoadenylates and their bacterial alkaline phosphatase (BAP)-resistant cores (which do not contain triphosphate groups) were able to inhibit protein synthesis in hypertonically permeabilized cells, whereas in cell-free system these BAP-treated nucleotides were inactive [7]. In another case pppA2’p5’A2’pSfA co-precipitated with calcium phosphate showed inhibitory activity on protein synthesis in a variety of different cell types [12,13]. In [14], inhibition of DNA synthesis by (2’ + 5’)-oligoadenylates was observed in lymphocytes stimulated by Con A. Here, we report protein synthesis inhibition in intact mouse lymphocytes, hepatocytes and bone marrow cells by exogenous A2’p5’A, A2’p5’A2’p5’A and its bis-phosphoramidate analog.

Effects of adenosine 5'-phosphate esters with lipoid hydroxy compounds (adenosine nucleolipids) on the activity of enzymes of cyclic AMP system.

It has been shown by several investigators that crude preparations of adenylate cyclase, exposed to higher concentrations of several hydroxy compounds, form corresponding esters of adenosine 5’-phosphate [l-6 1. Esters of this type might be theoretically formed in vivo after ethanol digestion or from glycerol released during the lipolysis [6,7 J . The physiological significance of these compounds, however, remains undefined. Since monoand di-glycerides are formed as intermediate products duringlipolysis we have synthetized, as further theoretically possible alcohol esters of AMP formed in the body, the adenosine S’esters of glycerol-monooleate, glycerol-monostearate and further lipoid residues containing drugs. We report here some representative results following the effects of these compounds (adenosine nucleolipids) on the activity of enzymes of cyclic AMP system.

Caesium fluoride-induced changes in the c.d. spectra of synthetic DNA fragments

Ten DNA fragments containing self-complementary alternating sequences of adenine and thymine differing in length and the starting nucleotide were studied by c.d. spectroscopy. It was found that d(TATATATA) but not d(ATATATAT), d(TATATA), d(CTATATAG) or (dT-dA)20 isomerized into the unusual X-DNA double helix at molar concentrations of CsF in solution. But in contrast to poly(dA-dT), the octamer (dT-dA)4, isomerized very slowly, at relatively low CsF concentrations and the isomerization was strongly dependent on the octamer concentration. A model is proposed to account for the observed properties of the B-to-X isomerization on the oligomer level.

Cloning and expression in Escherichia coli of the synthetic proenkephalin analogue gene

Enkephalins are pentapeptides with opioid activity that have been found in brain and other neural tissues. They are released by proteolytic processing of the proenkephalin, which contains several enkephalin sequences each flanked by pairs of basic amino acid (aa) residues. We have constructed an artificial variant of the proenkephalin gene by concatenation of synthetic oligodeoxynucleotides (oligo) coding for Met-enkephalin preceded by two arginines. One of the resulting structures, containing eleven enkephalin sequences separated by pairs of arginine codons, was cloned in the expression vector pRE31. The biological activity of enkephalin was detected after the digestion of the isolated plasmid-coded protein with trypsin and carboxypeptidase B. The product of the synthetic gene may thus serve as a defined simplified substrate for the study of the not yet fully understood enzymatic mechanisms of proenkephalin processing.

Stability of 2′ – 5′)oligoriboadenylates in various sera

Avian and mammalian sera were found to contain an enzyme activity degrading 2–5A oligonucleotides. The most extensive degradation of the A2′p5′ A was observed in chicken serum. Degradation of this compound is not affected by the presence of cAMP, dsRNA, Mg2+, but is significantly inhibited by EDTA. The enzyme activity described is not inactivated by heating to 56°C for 30 min. The 5‐mU3′p5′ A has also been degraded in chicken serum