A Specific Modification Next to the Anticodon of Phenylalanine Transfer Ribonucleic Acid

Abstract

By mild treatment of tRNA;:& with acid (pH 2.9, 37", 3-4 hours) the base Y' , which according to RajBhandary, Khorana et al. is located right next to the anticodon, can be excised without breaking the chain. Y+ and the acid treated tRNAPhe (= tRNA&E) were characterized and the conversion of tRNAPhe to tRNAP,h,9 was investigated in detail.1. Yf is a rather hydrophobic compound of unknown structure. Y+ and ribose were found when the nucleoside Y was treated with acid. Some physical and chemical properties of Yf are reported. Yf can be isolated easily in pure form from oligonucleotides, tRNA:,h,e,,, or even unfractionated tRNAyeast (not tRNA,,l() by mild acid treatment and subsequent extraction into chloroform.2. tRNAgEl and tRNAPhe have very similar molecular sizes and shapes since their Sephadex elution profiles were overlapping. The facile separation of tRNAPhe and tRNA&El by countercurrent distribution, on columns of methylated albumin on Kieselgur or benzoylated DEAEcellulose is due, a t least in part, to the removal of a hydrophobic residue from an exposed region of the tRNAPhe.3. tRNA&g can be charged with Phe to the same extent as tRNAPhe. Since also the Michaelis constant of phenylalanyl tRNA synthetase is the same for both tRNAs (1.5 x lo-' M) and the maximal velocity of charging is decreased by only one third in tRNAGE, it can be concluded, that Y and probably the whole anticodon loop are not essential parts of the synthetase recognition region of tRNAPhe in homologous charging reactions. 4.It was found that tRNAgE and tRNAPhe from which the -CpCpA endgroups had been removed do not differ in their acceptor capacities for CTP and ATP.5 . I n the ribosome systems, Phe-tRNAP,h,", does not bind to poly (U) or other polynucleotides and does not transfer its amino acid onto growing peptide chains. The complete loss of codon recognition is attributed largely to a change in the conformation of the anticodon loop. The ultraviolet spectra of an Y-containing hexanucleotide and of its acid conversion products are in agreement with the notion of a strong interaction between Y+ and its neighbouring bases, as are the rate differences in the liberation of Yf from Y 111.5 times faster] and the hexanucleotide, respectively.6. The rate of tRNAEz formation, as measured by methylated albumin on Kieselgur-column chromatography and binding assays, is the same as the rate of Y+-liberation from the hexanucleotide. The acid induced conversion of tRNAPhe to tRNAP,",", is a completely selective reaction affecting Y as the only nucleoside of tRNAPhe.

Complementary oligonucleotide binding to yeast tRNA PheHCl

“…In other words, the presence of the modified purine, adjacent to the anticodon, is important for a stable codon-anticodon interaction. It has been reported that tRNAE:I cannot form a stable complex with mRNA on the ribosome [9]. This observation apparently reflects the weak codontRNA?G, interaction reported in table 2, which cannot be enhanced or restored by the ribosome.…”

“…Association constants were determined by equilibrium dialysis experiments [5,7,8,IO]. Oligomer concentrations were in the range of 0 1-O 3 /.LM treatment'of tRNAphi tRNAPhe HCI was prepared by acid as described by Thiebe and Zachau [9]t. Association constants were classified as previously discussed [7, lo].…”

“…Recently, the technique of complementary oligonucleotide binding has been developed to explore the structure of tRNA [5-81. We used this technique in order to explore structural changes induced by an excision of the base Y [9], which is adjacent to the anticodon of yeast tRNAPhe. The results presented in this communication…”

Origin of the nucleoside Y in yeast tRNAPhe

“…The results of two experiments with different yeast strains and reversed label are presented in table 1. The values for Y+ were calculated with ~tn e237n m of 3 × 104 M -1 • cm -1 [6]. In both experiments no significant radioactivity was found in U and C. The very good coincidence between the specific radioactivity of Y+ and G, in contrast to that of A makes it clear that Y is biosynthetically derived from G.…”

“…After the base Y+ had been isolated by treatment with acid and extraction with chloroform [6] tRNA was split with T2 RNAase and the products were separated first by paper electrophoresis at pH 3.5.The material from the spots containing Ap, Gp, Up, and Cp was then treated with alkaline phosphatase and rechromatographed on paper. Y+ was also purified by chromatography in the same system.…”