Date of Award

1980

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Abstract

A tRNA (adenine-1)-methyltransferase from wheat germ was partially purified 9.6-fold and used to methylate tRNA('Phe) from E. coli. The site of methylation was determined to be the invariant adenine in the T(psi)C loop as revealed by sequence analysis of a pancreatic RNase digest, post-labelled with {(gamma)-('32)P}ATP. The elucidation of this site was preceded by studies on the enzyme itself, with bulk E. coli tRNA used as the substrate. Enzyme optima were determined for pH, temperature, {putrescine} and {magnesium}. Respective values were 7.5, 37(DEGREES)C, 20 mM, and an inactivation as the magnesium concentration was increased. Kinetic parameters at pH 7.5 were also obtained for this enzyme. The Km for S-adenosyl-L-methionine was 1.5 (mu)M, and for tRNA was 1.8 (mu)M. The Ki values for S-adenosyl-L-homocysteine for varied levels of tRNA and S-adenosyl-L-methionine, respectively, were 37 (mu)M and 3.7 (mu)M. This work has implications regarding the effects of characterized in vitro methylation of heterologous tRNAs on their biological function. Another aspect of this work concerned the sequence analysis of Euglena gracilis cytoplasmic tRNA('Phe). This sequence was determined via in vitro labelling of partial and complete nucleolytic digests of the intact tRNA with {(gamma)-('32)P}ATP. The labelled fragments derived thereof were resolved and individually sequenced via a combination of chemical, electrophoretic, and chromatographic techniques. This sequence provides indirect evidence for the endosymbiotic theory of evolution of chloroplasts. The Euglena gracilis chloroplastic tRNA('Phe) {Chang et al., Cell, 9, 717 (1976)} is prokaryotic in comparison to this and other prokaryotic tRNAs('Phe). The sequence of the cytoplasmic tRNA('Phe) is: pG-C-C-G-A-C-U-U-A-m('2)G-C-U-Cm-A-G-D-D-G-G-G-A-G-A-G-C-m('2)(,2)G-(psi)-(psi)-A-G-A-Cm-U-Gm-A-A-Y-A-(psi)-C-U-A-A-A-G-m('7)G-U('*)-C-C-C-U-G-G-T-(psi)-C-G-m('1)A-U-m('5)C-C-C-G-G-G-A-G-(psi)-C-G-G-C-A-C-C-A.

Pages

186

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