A CHANGE IN THE SPECIFICITY OF TRANSFER RNA AFTER PARTIAL DEAM1INATION WITH NITROUS ACID By JOHN CARBON AND JAMES B. CURRY MOLECULAR BIOLOGY DEPARTMENT, ABBOTT LABORATORIES, NORTH CHICAGO, ILLINOIS Communicated by Paid Berg, December 18, 1967 The rapid advances in the structural chemistry of tRNA during the past few years have offered firm support to the concept of a triplet anticodon in tRNA which pairs with a triplet codeword on the RNA message.' This anti- codon-codon combination is thought to depend on the usual Watson-Crick base- pairing for specificity, although it is recognized that the specificity patterns are somewhat relaxed in the third or "wobble" position of the triplet,2 and that ribosomal interactions are important in maintaining a high degree of fidelity.3 Changes in the specificity of tRNA observed in strains of E. coli carrying certain suppressor mutations are thought to arise either by mutations at or near the anticodon or amino acid recognition sites of a particular tRNA.4 Recently the amber su +I mutation has been shown to occur in the gene speci- fying a tRNAtyr, leading to a change in the 5'-base of the anticodon.A If the alteration of a single base of an anticodon is sufficient to change the specificity of tRNA, then the in vitro random deamination of tRNA should lead to a population of molecules with altered specificity. For example, subjection of tRNAGGA with the presumed anticodon UCC to random nitrous acid deamin- ation should yield molecules containing the anticodons, UCU and UUC (Fig. 1). FIG. 1.-Possible anticodon changes in tRNAGly (Glu) (Gly) (Arg) produced by in vitro deamination with nitrous acid. CODONS: - GAA - - GGA - - AGA - Codons are shown with the 5'-end on the left; anticodons, I.: with the 5'-end on the right. It is assumed that the ANTICODONS: CUU HN02 CCU HN0, UCU anticodon of tRNAG% is as shown. Only single base changes are considered. According to current theory, such altered species would insert glycine into polypeptides in response to the arginine or glutamic acid codewords, AGA and GAG. This assumes that such altered molecules would still interact normally with the glycine activating enzyme (see Discussion). Since nitrous acid deamination of tRNA is a random process which inactivates tRNA after only two to three hits per molecule,6 active molecules suffering anticodon hits will be present in minute concentration in HNO2-treated tRNA. The availability of an extremely sensitive in vitro assay7 to measure incorpora- tion of C14-glycine in response to the codewords AGA and GAG has now made it possible to demonstrate one of the predicted specificity changes in tRNAgA produced by HNO2 deamination (Fig. 1). This assay depends upon the use of the alternating polyribonucleotide, poly (AG),* containing only the codewords AGA and GAG and normally specifying the synthesis of only an arginine-glu- tamic copolypeptide using an in vitro system derived from E. coli.8 This assay was previously used to demonstrate that tRNA from E. coli strains carrying the missense suppressor mutation su+ will introduce C14-glycine into an alternating 467 Downloaded by guest on September 24, 2021 N. A. S. 468 BIOCHEMISTRY: CARBON AND CURRY PROC. copolypeptide with glutamic acid.7'9 We now show that E. coli B tRNA preparations enriched in tRNAcIGA, but free of tRNAargA, will support such poly (AG)-dependent C"4-glycine incorporation after treatment with nitrous acid under controlled conditions. Materials and Methods.-Transfer RNA was purchased from the General Biochemicals Co. or was isolated from E. coli by the method of Zubay,10 except that the preparations were treated with 0.2 M Tris (unneutralized) for 15 min to hydrolyze aminoacyl tRNA. Silicic acid (325 mesh, suitable for chromatography) was obtained from the Fisher Scien- tific Co. This material was prewashed with 1 M HCl and then water until neutral, and the fines were removed by decantation. Labeled amino acids were purchased from the New England Nuclear Corp. E. coli strains HfrRtryB- and HfrR- sut were obtained from P. Berg, Stanford University. Poly U, C, and A were from Miles Chemical Co. Random poly AG (7.4:1) and poly UG (3:1) were prepared using M. lysodeikticus poly- nucleotide phosphorylase, purified to stage VI by the method of Thanassi and Singer." The in vitro assay for the poly (AG)-dependent incorporation of C'4-glycine into TCA- tungstate insoluble material was carried out as described previously,7 using prewashed ribosomes and a protamine-treated 100,000 X g supernatant prepared from E. coli strain HfrRtryBj,12 The poly (AG)-dependent incorporation C14'arginine alternating copolypeptide with L-glutamic acid was measured in a similar manner,8 except that the 100,000 X g supernatant was rendered virtually tRNA-free by either a pre- liminary DEAE-cellulose treatment'3 or by protamine treatment to A2W/A2W = 1.5.7 The alternating polydeoxyribonucleotide, d(AG:TC), was prepared by replication of an authentic samplel4 (originally from H. G. Khorana) with DNA polymerase (fraction VII of Richardson et al.'5). RNA polymerase was purified to stage IV by the procedure of Chamberlin and Berg.16 Methylated albumin on silicic acid (MASA) chromatography: Prewashed silicic acid (70 gm) was suspended in 0.05 M Na phosphate buffer (pH 7.0), boiled briefly, cooled, and a solution of 2 gm methylated albuminl7 in 100 ml water added with stirring. Excess albumin was removed by decantation and suction filtration. The filter cake was sus- pended in a solution of 0.05 M sodium acetate buffer (pH 5.4)-0.3 M NaCl and packed into a 2 X 31-cm column. After equilibration with 2-3 column volumes of the same buffer, 100 mg of E. coli B tRNA in 20 ml buffer was applied to the column. Elution was carried out using a linear gradient to 1.0 M NaCl (2000 ml total volume) in the same buffer, collecting 10-ml fractions. Similar columns (4.4 X 33 cm) were run using 500 mg tRNA and the same total gradient volume with only a slight loss in resolution. Aliquot samples were assayed for C'4-glycine and C14-arginine acceptor ability in the usual manner.'8 The fractions were pooled in groups of 10-20 tubes, and the tRNA precipitated by adding two volumes of ethanol. After isolation by centrifugation, the pellets were washed with ethanol and ether, vacuum-dried, and stored frozen in aqueous solution. Nitrous acid deamination of prefractionated tRNA: The MASA pools were treated with nitrous acid as previously described,6 except that 0.8 M acetate buffer (pH 4.40) was used and the reaction mixture was not maintained at constant pH with an automatic titrator. Polynucleotide-stimulated binding studies: C'4-glycyl tRNA was prepared as previously described.'9 Ribosomal binding was measured by method B of Sl11 et al.,'9 except that the incubation was for 10 min at 37°. Ribosomes were prewashed twice with buffered 3 M KCl by the method of Smith.20 Results.-Any specificity changes induced by nitrous acid deamination of tRNA could be due to (1) a change in or near the anticodon of a tRNA, causing a change in the normal codon-anticodon relationship (Fig. 1), or (2) a change in the aminoacyl tRNA synthetase recognition site, or conformation of the tRNA, such that an incorrect amino acid is esterified onto the terminal nucleotide. In the particular case described here, incorporation of glycine into polypeptides Downloaded by guest on September 24, 2021 VOL. 59, 1968 BIOCHEMISTRY: CARBON AND CURRY 469 in response to the arginine codeword, AGA, could result from an anticodon deamination in tRNAGGA, or a change in tRNAAGA such that it accepts glycine instead of or in addition to arginine. These possibilities are identical to those described previously as possible changes in tRNA produced by the missense suppressor mutation designated S 36. 9 To determine which of these possi- bilities is pertinent in the studies described here, it was necessary to prefraction- ate the crude tRNA to separate tRNAGGA from tRNAAGA, and to individually subject tRNA containing these species to deamination. Prefractionation of E. coli B tRNA and localization of tRNA gGGA and tRNA A: Transfer RNA from E. coli B was fractionated over methylated albumin on silicic acid (MASA) columns, resulting in partial separation of the tRNAgly and tRNAarg (Fig. 2). After fractions were combined to form five pools (see Fig. 2), FIG. 2.-MASA column = 1 | 431 4 | 5 I chromatography of E. coli B Ilk 2A tRNA. See Methods section for x a description of the fractiona- Iva tion conditions. 30* 0-----, optical density at i00 1.6 260 m/L. l A % 7 60 *-4, C'4-glycine acceptor X ItA 2 O-O, C14-arginine acceptor X 400 ability. Fractions were pooled in five . 200 0.4 sections (arrows) for isolation of tRNA. 0 OaA -i0- 90 100 110 120 130 --140 110 120 *7 Tou n"r the tRNA was isolated and assayed for acceptor ability with C14-glycine and C'4-arginine. Note that pool 1 is free of arginine acceptor ability and is threefold enriched in tRNAgly over the unfractionated tRNA (Table 1). Pool 2 tRNA, although over twofold enriched in tRNAgly, is contaminated with a very small quantity of tRNAarg. In order to localize the tRNAglyA, as distinguished from the glycine-specific tRNA's responding to GGU, GGC, and GGG, samples of tRNA from pools 1-5 were charged with C14-glycine of high specific activity, and the binding2' to E. coli ribosomes was measured in the presence of the random polynucleotides, poly AG (7.4:1) and poly UG (3.0: 1).