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Combination of chemical and enzymatic methods for the synthesis of triribonucleoside diphosphates containing azaG and the stimulation of binding of [ 14 C]aminoacyl-tRNA's to ribosomes in the presence of these triplets are described. AzaG could replace guanosine in the 5′-position of codons for valine, alanine and aspartic acid as well as in the second position of codons for arginine. However, the template activity of azaG-containing triplets is lower than that of “normal” codons. The results with the GpUpG codon for valine showed that Escherichia coli tRNA and purified yeast [ 14 C]Val-tRNA 2 Val responded best to GpUpG, less well to azaGpUpG even less well to GpUpazaG and only slightly to azaGpUpazaG. The possible mechanism of the inhibition of protein synthesis by azaG in growing cells on the basis of lower template activities of azaG-containing triplets is discussed.

1. 1. 5-Azacytidine, in concentrations higher than 0.5 μg/ml, strongly inhibits the production of viable phage particles by bacteria infected with bacteriophage T4. The inhibitory action can be abolished completely by cytidine and mitigated slightly by deoxycytidine. The inhibition becomes irreversible about 3 min after the addition of 5-azacytidine. When added later than 12 min after infection, 5-azacytidine does not interfere with phage maturation. 2. 2. Synthesis of RNA is not affected by 5-azacytidine but synthesis of DNA is strongly inhibited. The inhibition of DNA synthesis cannot be reversed by deoxy-cytidine, but is reversed by cytidine. In un-infected cells 5-azacytidine does not inhibit the synthesis of DNA. 3. 3. When 5-azacytidine is added immediately after infection, total protein synthesis is inhibited only in the late period of phage development; the synthesis of late phage-specific proteins is inhibited, whereas the early enzymes are slightly over-produced. 4. 4. The frequency of genetic recombination is not affected by 5-azacytidine.

1. 1. When 5-azacytidine is added to a culture of Escherichia coli induced to synthesize β-galactosidase, the synthesis of the enzyme slows down immediately and comes to complete rest about 12 min after the addition of the inhibitor. Upon addition of either cytidine or uridine, the synthesis of the enzyme is resumed at full or reduced speed, depending on the time of action of 5-azacytidine. 2. 2. Total protein synthesis is inhibited by 5-azacytidine to about 6 % of the control. Synthesis of RNA, as measured by pulse labelling with adenosine or uridine, is slightly inhibited. General inhibition of RNA synthesis is never observed. 3. 3. 5-Azacytidine does not inhibit the synthesis of DNA, as measured by incorporation of thymidine. Whereas in a normally growing culture the incorporation of thymidine soon ceases as a result of conversion of thymidine to thymine by thymidine phosphorylase, in the culture treated with 5-azacytidine the incorporation of thymidine proceeds indefinitely at a constant rate. The synthesis of inducible thymidine phosphorylase is inhibited by 5-azacytidine, whereas the action of the pre-formed enzyme is not affected. 4. 4. In a culture containing 5-azacytidine an unbalanced growth is observed, resulting from concurrent inhibition of protein synthesis, cessation of cell division and growth and continued synthesis of DNA. 5. 5. It is concluded that 5-azacytidine inhibits protein synthesis by selectively affecting the function of some species or portion of RNA essential for protein synthesis.