Base substitutions in the wobble position of the anticodon inhibit aminoacylation of E. colitRNAfMet by E. coli Met-tRNA synthetase

Abstract

Derivatives of E. coli tRNAfMet containing single base substitutions at the wobble position of the anticodon have been enzymatically synthesized in vitro. The procedure involves excision of the normal anticodon, CAU, by limited digestion of intact tRNAfMet with RNase A. RNA ligase is then used to join each of four trinucleotides, NAU, to the 5' half molecule and to subsequently link the 3' and modified 5' fragments to regenerate the anticodon loop. Synthesis of intact tRNAfMet containing the anticodon CAU by this procedure yields a product which is indistinguishable from native tRNAfMet with respect to its ability to be aminoacylated by E. coli methionyl-tRNA synthetase. Substitution of any other nucleotide at the wobble position of tRNAfMet drastically impairs the ability of the synthetase to recognize the tRNA. Measurement of methionine acceptance in the presence of high concentrations of pure enzyme has established that the rate of aminoacylation of the AAU, GAU and UAU anticodon derivatives of tRNAfMet is four to five orders of magnitude slower than that of the native or synthesized tRNA containing C as the wobble base. In addition, the inactive tRNA derivatives fail to inhibit aminoacylation of normal tRNAfMet, indicating that they bind poorly to the enzyme. These results support a model involving direct interaction between Met-tRNA synthetase and the C in the wobble position during aminoacylation of tRNAfMet.

Anticodon loop size and sequence requirements for recognition of formylmethionine tRNA by methionyl-tRNA synthetase.

“…On the basis of the ratio of 32P to A2w, specific activities of 800-1,000 pmol/A2m unit are obtained. As noted previously (3), similar specific activities are obtained for equivalent amounts of intact tRNAfmet after electrophoresis and isolation from polyacrylamide gels due to contamination of small samples with nondialyzable, ethanol-precipitable UV-absorbing material from the gels. The 32p label incorporated during the synthesis has therefore been used to calculate the actual concentration-of each product for aminoacylation studies.…”

“…These results focused our attention on the anticodon, the variable loop, and the acceptor stem of tRNAfMet for more detailed analysis of the structural requirements for protein-tRNA recognition. We have shown that the anticodon wobble base plays an essential role in this process (3,4 Synthesis of Oligonucleotides. The trinucleotides GpCpA and CpApU were synthesized by reaction of GpC with ADP and CpA with UDP, using polynucleotide phosphorylase as described by Thach and Doty (7).…”

“…These results focused our attention on the anticodon, the variable loop, and the acceptor stem of tRNAfMet for more detailed analysis of the structural requirements for protein-tRNA recognition. We have shown that the anticodon wobble base plays an essential role in this process (3,4). In 37°C) of poly(A,C) (1:1) with RNase U2 (0.5 unit/mg of RNA) in 50 mM sodium acetate, pH 4.5, followed by incubation with 0.125 M HCI at room temperature for 6 hr.…”

“…The tetranucleotide CpApUpAp was synthesized by addition of pAp to CpApU, using RNA ligase. All oligonucleotides were purified by column chromatography and analyzed as described elsewhere (3). Oligonucleotides were phosphorylated at the 5' terminus by using [y-32P]ATP and PseT 1 polynucleotide kinase (9).…”

“…Intact molecules containing normal and altered anticodon loop sequences were synthesized by using RNA ligase and polynucleotide kinase,_ and the 3'-terminal sequence was enzymatically replaced by using tRNA nucleotidyltransferase, as described before (3 cpm/pmol). Samples were equilibrated at 260C for 5 min and reactions were initiated by addition of purified E. coli Met-tRNA synthetase.…”