Guanosine Triphosphate Dependent Enzymic Binding of Aminoacyl Transfer Ribonucleic Acid to Yeast Ribosomes

Abstract

The binding of phenylalanyl-tRNA to purified yeast ribosomes at low magnesium concentrations is dependent upon one of the yeast supernatant transfer factors (factor A). This reaction requires GTP. The GTP analogue guanylyl methylene diphosphonate cannot be substituted for GTP but it acts as a good inhibitor. After incubation of the ribosomes with phenylalanyl-tRNA, factor A and GTP practically no compound other than phenylalanyl-tRNA itself is found linked to the ribosomes.I n addition to this enzymic type of binding, phenylalanyl-tRNA is also bound to yeast ribosomes in the absence offactor A and GTP provided that the magnesium concentration is increased to about 20 mM. This non-enzymic binding is effectively inhibited by deacylated tRNA and this inhibition is prevented if factor A and GTP are included in the incubation mixtures.The binding of N-acetylphenylalanyl-tRNA to yeast ribosomes also requires GTP, and is dependent upon factor(s) other than the amino acid transfer factors, which are bound t o the ribosomes.Evidence obtained with an Escherichia coli system has shown that the binding of the initiator N-formylmethionyl-tRNA to the ribosome-template complex is mediated by initiation factors in a reaction which requires GTP [I-61. Whether the binding of the aminoacyl-tRNAs involved in the elongation of the peptide chain is enzymically mediated or not has been a point of discussion during the last few years. Schweet and his group fist reported that the binding of phenylalanyl-tRNA to rabbit reticulocytes ribosomes requires one of the amino acid transfer factors (TF,) and GTP [7]. A number of efforts to show this dependence with other systems were unsuccessful. While this work was in progress it has been reported that the binding of non initiator aminoacyl-tRNAs to E. coli [8,9] and rat liver [lo] ribosomes is also mediated by one of the amino acid transfer factors in a GTP dependent reaction.I n a previous paper we reported that 'yeast ribosomes can effectively bind phenylalanyl-tRNA a t high magnesium concentrations in the absence of supernatant transfer factors and GTP [ll]. After the resolution of the yeast amino acid transfer system into complementary factors [12-141 we made further efforts to elucidate the role of these transfer factors in amino acid polymerization. We have now found U n w l abbreviations. PPO, 2,5 diphenyloxazole; POPOP, 1,4-bis-2-(5-phenyl-oxazolyl)benzene; GMP-PCP, 5'-guanylyl-p y -methyhe diphosphonate, tRNA, transfer RNA, phenylalanyl-tRNA, charged tRNA carrying phenylalanyl residue. that a t magnesium concentrations within the physiological range the poly U directed binding of phenylalanyl-tRNA t o yeast ribosomes is dependent on one of the yeast amino acid transfer factors and GTP. I n contrast with these findings, neither of the amino acid transfer factors are involved in the binding of N-acetylphenylalanyl-tRNA to yeast ribosomes. Our results suggest that N-acetylphenylalanyl-tRNA is bound to yeast ribosomes through the action of some other factor(s), presumably initiation fact...

N‐Acetylphenylalanyl‐tRNA Hydrolase from Yeast

“…Acetylation of the ['4C]amin~acyl-tRNAs was performed using acetic anhydride [lo]. The extent of acetylation was checked by alkali treatment of the acetylated [14C]aminoacyl-tRNA followed by paper chromatography [9]. In all cases more than 95% acetylation was achieved.…”

“…Crude yeast tRNA and tRNAPh" from yeast and Escherichia coli were purchased from Boehringer. The charging of tRNA with radioactive amino acids and the isolation of the [14C]aminoacyl-tRNAs was done as described elsewhere [9]. The specific activities of the different amino acids were as follows: phenylalanine, leucine and valine, 140 Ci/mol; methionine, 170 Ci/mol.…”

“…[15]. Analysis of the resulting compound [9] showed that more than 90 % of the aminoacyl-tRNA was benzoylated.…”

“…A control containing deacylated tRNAPhe was processed in the same way. Aliquots of the two preparations were tested for their capacity to accept ['"Clphenylalanine and [14C]valine in the standard reaction mixtures for the charging of tRNA [9]. A soluble protein fraction from yeast (105000 x g supernatant filtered through Sephadex G-25) was used as source of the phenylalanyltRNA and valyl-tRNA synthetases.…”

“…The preparation of this hybrid molecule was performed according to the method described [16] [9] showed that at least 95 % was acetylated. The identification of the tRNAPh' in the hybrid molecule was made by recharging the tRNA resulting from alkaline hydrolysis of the N-acetyl-['"C]valyl-tRNAPh" preparation, after oxidation with periodate.…”