Phenylalanyl-tRNA synthetase from baker's yeast: role of 3'-terminal adenosine of tRNA-Phe in enzyme-substrate interaction studied with 3'-modified tRNA-Phe species.

Abstract

ABSTRACTtRNAPhe species from baker's yeast modified at the 3'-terminus in many cases are phenylalanylatable substrates. Out of several tRNAPhe species possessing a modified 3'-end that cannot be phenylalanylated, only two, tRNAPheC-C-2'dA and tRNAPheC-C-formycinoxi-reds are strong competitive inhibitors for tRNAPheC-C-A during phenylalanylation. In the ATP/PPi exchange, both these inhibitors reduce Vman, to about 25%o; but whereas tRNAPheCC_2'dA has no influence on Km ATP and Km Phe during ATP/PPj exchange, tRNAPheCC-formycinox i-red reduces Km AT! from 1430 IAM, found in the absence of tRNAPhe, to 230 jiM, and Km Phe, from 38 to 14 uM. The values found in the presence of tRNAPheC-C-CformycinOxi-re during ATP/PPi exchange, are identical with those determined in the phenylalanylation of tRNAPheC_C-A.All other tRNAPhe species carrying a modified 3'-end that cannot be phenylalanylated exhibit a mixed competitivenoncompetitive inhibition in the phenylalanylation reaction. In the ATP/PPi exchange, they do not influence Km ATP and K,, Phe and only weakly, if at all, Vmax.The results show that the 3'-adenosine of tRNAPhe cannot solely be a passive acceptor for phenylalanine, but must in addition play an active role during enzyme-substrate interaction. The data can be consistently explained by the hypothesis that the 3'-adenosine of tRNAPhe triggers a conformational change of the enzyme.In the living cell part of the genetic information stored in the DNA is translated into proteins. This is achieved by first transcribing the information into mRNAs and then translating the latter into peptide sequences on the ribosome. Prior to translation on the ribosome, the amino acids are activated by attachment to the 3'-terminal ribose of their particular tRNAs.The overall aminoacylation reaction is achieved according to tRNAaa + ATP + aa + E 2 aa-tRNAaa + AMNP + PPj + E, where E is the enzyme and aa is the amino acid. This reaction can be followed experimentally by the incorporation of radioactively labeled amino acid into tRNA.

Interrelation between Transfer RNA and Amino‐Acid‐Activating Sites of Methionyl Transfer RNA Synthetase from Escherichia coli

“…Therefore, 3'-terminal adenosine binding to methionyl-tRNA synthetase is not a pre-requisite condition for the triggering of inhibition of the ATP-PPi exchange by tRNA. At this stage, a different mode of behaviour of yeast phenylalanyltRNA synthetase has to be noted [12]. The modified tRNAPhes lacking the terminal adenosine are reported not to interfere with the phenylalanine-activating site, while the tRNAPh' having a modified or a formycine-substituted terminal adenosine does interfere.…”

Distinct Steps in the Specific Binding of tRNA to Aminoacyl-tRNA Synthetase. Temperature-Jump Studies on the Serine-Specific System from Yeast and the Tyrosine-Specific System from Escherichia coli

“…The tRNA must be bound in such a way that aminoacyl transfer is feasible. If the appropriate juxtaposition of tRNA and activated amino acid is not achieved with the recombination step a consecutive reaction is necessary to orient the tRNA properly on the enzyme surface (cfi [20]). It seems to be an attractive speculation to regard the observed second reaction as necessary for this orientation.…”

Functional group recognition at the aminoacylation and editing sites of E. coli valyl-tRNA synthetase

“…Altering the 3Ј-terminal adenosine (Best and Novelli 1971;Tal et al 1972;Rether et al 1974;Von der Haar and Gaertner 1975) or its ribose moiety (Uziel and Jacobson 1974) results in complete loss or considerable decrease in aminoacylation efficiency, largely due to a decrease in k cat . 3Ј-end-modified tRNAs are also defective in editing (Tardif et al 2001) and are readily misacylated (Tamura et al 1994;Tardif et al 2001;Nordin and Schimmel 2002).…”