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Studies of inhibition of aminoacyl-tRNA synthetases Roy, Kenneth Leo Joseph

Abstract

The structural features involved in recognition of transfer ribonucleic acids (tRNAs) by aminoacyl-tRNA synthetases have been investigated using an inhibition assay. It was expected that any oligo- or polynucleotide possessing any of the structural features required of a tRNA for recognition by its aminoacyl-tRNA synthetase would cause specific competitive inhibition of that enzyme. Oligonucleotides from a pancreatic r ibonuc lease digest of RNA were separated according to chain length, then certain isoplithic fractions were subfractionated on the basis of composition. A number of the separated oligonucleotides caused inhibition of valine, lysine and arginine estexification, but in no case was this specific for a given amino acid or kinetically competitive . Oligonucleotides from a ribonuclease T₁ digest of yeast tRNA were separated into isopLithic fractions , but none of these, were significantly inhibitory toward valine, lysine, aspartic acid or phenylalanine esterification to tRNA at the levels tested. Oligonucleotides from a pancreatic ribonuclease digest of poly A and poly U, when separated into isoplithic fractions, did not competitively or specifically inhibit lysine or phenylalanine esterification to tRNA. A number of studies were conducted to determine whether or not periodate-oxidized tRNA (tRNAox) could be used as a starting point for recognition studies. Attempts were made to find conditions under which inhibitory samples of tRNAox could be partially digested with nucleases but still retain the capacity to inhibit aminoacylation. These experiments gave no indication of success and so were abandoned. Cyanoethylated tRNA was oxidized with periodate to determine whether or not it had the capacity to inhibit, but the results were inconclusive. Samples of tRNA were subjected to stepwise degradation and a collection of degraded species was obtained. These were found to vary in their capacity to inhibit valine, lysine and phenylalanine esterification to tRNA. Thus, none of the degraded species inhibited phenylalanine esterification, whereas all of those prepared inhibited valine esterification. Lysine esterification had an intermediate response to these tRNAs. The necessary control experiments were carried out to show that the reagents used in the degradations had no effect on acceptor activities. The response of valine and phenylalanine esterification to variations in magnesium ion concentrations was measured and it was shown to differ significantly for each amino acid. Phenylalanine esterification was shown to be strongly inhibited by excessive quantities of magnesium ion while valine esterification was not. A need for a quantity of magnesium ion in excess of that amount chelated by adenosine triphosphate was demonstrated. Attempts were made to measure the extent of binding of magnesium ion to tRNA under the conditions of aminoacylation assays by the equilibration dialysis technique. However, the errors of the method used for magnesium ion determination were too great to allow any firm conclusions to be drawn.

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