Title

Conformational Properties of Polyribonucleic Acids: Studies with Yeast Transfer-RNA, Synthetic Homopolyribonucleotides and Rabbit Globin Messenger-RNA

Date of Award

1980

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biology

Advisor(s)

John Vournakis

Keywords

Biopolymers, Ethidium bromide, Conformation, Magnesium, Environmental factors

Subject Categories

Biophysics

Abstract

The effect of various physical parameters on the conformation of three classes of biopolymers in solution, was investigated under equilibrium conditions. Using ethidium bromide as a probe, a description of both the conformational properties and conformational transitions occurring in transfer RNA became possible. A novel class of interactions between ethidium bromide and tRNA was revealed. This interaction occurred when the probe reacted with a solution of tRNA containing 5mM magnesium ions. Such an interaction can be considered as a useful index for the occurrence of tertiary interactions in tRNA in solution.

The effect of magnesium and manganese ions on the conformation of the hydrogen bonded hybrids polyribocytidylate(.)oligodeoxyguanylate and poly2'-O-methylcytidylate(.)oligodeoxyguanylate was studied. The results were compared with the ability of the hybrids to serve as efficient substrates for avian myeloblastosis virus DNA polymerase under the same solution conditions. It was found by this comparison that the conformation of these substrates as affected by the aforementioned ionic species constitutes an important parameter controlling enzymatic activity.

The effect of environmental factors (such as heat, pH, ionic composition, etc.) as potential determinants of the conformation of rabbit globin messenger RNA (mixture of two messenger RNA species) was investigated. Findings provided evidence for the occurrence of major conformational changes in mRNA. From a macroscopic viewpoint both acidic pH and magnesium ions strongly enhanced the conformation and stability of the message.

The conformation of this particular class of biological macromolecules was studied in further detail, at the nucleotide level, for each mRNA species. The most important finding from this study was the strong susceptibility, to the single strand specific enzymatic probe used in these experiments, of the initiator region in (beta) globin mRNA as opposed to the very limited susceptibility of the analogous region of (alpha) globin mRNA by the enzyme. This finding can provide a molecular interpretation to the more efficient initiation of protein synthesis by rabbit (beta) globin mRNA chain than by rabbit (alpha) chain.

A search for 18s ribosomal RNA binding sites in the 5'-non coding region of rabbit globin mRNA was undertaken in an attempt to reveal similarities in the mode of initiation of protein biosynthesis among eucaryotic and procaryotic systems. Results indicated an absence of interacting sequences with 18s rRNA in the 5'-non coding region of (beta) globin mRNA. This result is consistent with the lack of protection against enzymatic hydrolysis of eucaryotic mRNA sequences in a procaryotic ribosome binding system observed by other investigators, and reveals one more possible difference between procaryotic and eucaryotic mechanisms of initiation of protein synthesis.

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