29S H. G. BOMAN, I. A. BOMAN 



Structurally related to arginine have been investigated and their effect 

 on the rate of formation of arginine-RNA will be described. In addition 

 cells obtained from different states of repression will be compared in 

 regard to the arginine-activating enzyme and the arginine-RNA. In 

 particular this comparison involves a mutant in which arginine no longer 

 represses enzyme formation. 



Methods and Materials 



The strains used were E. coli Hfr 30S0 (" wild type ") and Hfr 30S0A5, 

 a canavanine-resistant mutant [6] in which arginine can no longer repress 

 the synthesis of some enzymes involved in its own biosynthesis (in the 

 following this mutant is designated arg.R"). 



Growth of the cells was carried out aerobically in two types of minimal 

 media, A and S-2, [7, 8] both supplemented with monosodium glutamate 

 (1-67 g./l.) and glucose (5 g./l.). The cells were harvested near the end 

 of the logarithmic growth phase, collected by centrifugation in a Sharpies 

 centrifuge, and stored as a frozen paste. 



The preparation of the acceptor RNAcan be summarized as follows [9] : 

 The procedure was designed to avoid large volumes and ultracentrifuga- 

 tions and is essentially a combination of steps taken from the methods of 

 Crestfield ei ah [10] and Kirby [11] to which a lanthanum precipitation 

 [12] has been added. It consists of a heating step which breaks up the cells, 

 a deproteinization with phenol, an alcohol precipitation to remove phenol 

 and low molecular weight material, an extraction of the precipitate with 

 I M sodium chloride to separate the acceptor RNA from the high molecular 

 weight RNA, and a lanthanum precipitation to separate the acceptor RNA 

 from polysaccharides. 



The assay of the arginine-RNA* was carried out in a volume of o • 5 

 ml. at pH 7-0. The concentration of the components were o-i m tris- 

 maleate buffer, 0-002 M ATP, 0-004 ^ Mg + + (added as MgO), 0-002 m 

 glutathione, 0-002 M fructose diphosphate, 0-28 mM L-[^^C]-arginine 

 with 5-93 X 10*' c.p.m./|umole, 15-20 jtxg. arginine-activating enzyme in 

 0-5 ml., and 5-25 optical units of RNA in 0-5 ml. The reaction mixture 

 was incubated at 37° for 15 min. Mixing of reagents and termination of the 

 reaction by the addition of 2 ml. of o-oi m lanthanum nitrate in 0-5 M 

 perchloric acid were done in an ice bath. After the reaction was stopped, 

 o ■ 2 ml. of 5% commercial yeast RNA was added as carrier. The precipitate 

 was washed twice with 2 ml. of cold 5",, trichloroacetic acid containing 



* The following abbreviations are used: RNA, ribonucleic acid; tris, tris- 

 (hydroxymethyl)aminomethane ; ATP, adenosine triphosphate; c.p.m., counts 

 per min. ; optical unit (o.u.), the amount of RNA which in i ml. gives an extinction 

 of I at 260 m/x; U.S. P., United States Pharmacopoeia; DEAE, diethylaminoethyl. 



