Structural Studies on Phenylalanine Transfer Ribonucleic Acid from Yeast with the Spectroscopic Label Formycin

Abstract

Formycinmonophosphate (FMP) has been incorporated in to the 3'-terminus of phenylalanine tRNA from yeast by tRNA nucleotidyl transferase. tRNAPhepCpCpF can be aminoacylated with the cognate synthetase exhibiting the same K , and an about 50-times lowered V compared to tRNAPhepCpCpA. Quantum yield of FMP fluorescence in tRNAPhepCpCpF is about four times lower than in the monomer. Comparative studies of tRNAPhepCpCpF and the oligonucleotide CpApCpCpF show that the differences between the fluorescence properties of tRNAPhepCpCpP and the monomeric formycin-nucleotide are due only t o stacking interactions and do not arise from the tertiary structure of tRNA. After periodate oxidation of the 3'-terminal ribose of tRNAPhepCpCpF the full fluorescence of formycin is reconstituted. This emphasises the important role of the intact ribose ring for stabilization of stacking units. Periodate oxidation of tRNAPhepCpCpF followed by borohydride reduction leads t o a tRNA that acts as competitive inhibitor in the aminoacylation reaction. This, together with its high quantum yield makes it a valuable tool in fluorescence spectroscopic studies of the aminoacylation reaction.

Isoleucyl-tRNA synthetase from baker's yeast and from Escherichia coli MRE 600. Discrimination of 20 amino acids in aminoacylation of tRNAIle-C-C-A(3'NH2)

“…Further, the preparations were tested with a crude mixture of aminoacyltRNA synthetases and 14C-labelled amino acids except isoleucine, yielding only background radioactivity. From these tRNA"' preparations the modified tRNA species tRNA1Ie-C-C-A(3'NH2) were prepared with the aid of nucleotidyltransferase from yeast by reaction of 3'-deoxy-3'-amino-ATP with tRNA"'-C-C and analyzed as described by Sprinzl et al [15], Cramer et al [16], Maelicke et al [17], Fraser and Rich [18] and Sternbach et al [19]. The preparations were also controlled by HPLC [14] and found to be free of tRNA"'-C-C-A as must be expected after the several steps of preparation and purification.…”

Fluorimetric Study of Yeast tRNAPheCCF in the Complex with Phenylalanyl‐tRNA Synthetase

“…The formycin-labeled tRNAs were prepared as previously reported [9]. The amino acid acceptance is 1.46 nmollA260 unit for tRNA$FF whereas tRNA$&oxi-red is a competitive inhibitor with respect to native tRNAPhe in the acylation reaction.…”

“…They showed a red shift of the absorption spectrum from N(1)H to N(2)H tautomer. On the other hand, Mqelicke et al [9] studied the modification of the fluorescence properties of formycin monophosphate upon periodate oxidation, followed by borohydride reduction. They showed that both the excitation and emission spectra of FMPo,i.,,ci were red-shifted with respect to those of FMP.…”

“…The tenfold increase of the quantum yield of formycin in t R N A E F [9] upon periodate oxidation could thus be explained by the combination of two factors: (a) a shift in the tautomeric equilibrium responsible for a 2.5-fold stimulation; indeed, as shown in (91, the periodate oxidation of formycin triphosphate leads to a similar increase of the quantum yield; (b) a destacking of the 3'-terminal formycin from the adjacent cytosine, responsible for a fourfold stimulation of the quantum yield; indeed, as shown in [9], the incorporation of formycin in the tRNA roughly leads to a fourfold reduction of the quantum yield.…”

On the Stereochemistry of Activation of Phenylalanine by Phenylalanyl‐tRNA Synthetase from Baker's Yeast

“…tRNA""' and tRNA1le were isolated by an analogous procedure. tRNAPhe-C-C-F,,i.,,d was prepared as published [8]. Phenylalanyl-tRNA, seryl-tRNA, valyltRNA and isoleucyl-tRNA synthetases were purified according to [9].…”