Date of Award

Summer 1997

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Committee Director

Mark S. Elliot

Committee Member

Frank J. Castora

Committee Member

Christopher J. Osgood

Committee Member

James H. Yuan

Abstract

The presence of the queuosine modification in the wobble position of tRNAasn, tRNasp, tRNAhis, and tRNAtyr is associated with a decrease in cellular growth rate, an increase in the ability to withstand environmental stress, and differentiation of pleuripotent cells into mature phenotypes. The loss of this normal modification is strongly correlated with neoplastic transformation and tumor progression of a wide variety of cancers.

The "normal" system for formation of the queuosine modification in tRNA was studied in human fibroblast cell cultures and in mouse, rat and human liver tissues. The queuosine modification system is shown to be made up of three distinct mechanisms: uptake of the queuine base across the plasma membrane; incorporation of this base into cytoplasmic tRNA; and salvage of the base from products of normal tRNA degradation. The queuine membrane transporter and incorporation enzyme are activated via phosphorylation by protein kinase C and inactivated by the action of a phosphatase. This regulation by phosphorylation integrates the queuosine modification system into a very sensitive eukaryotic cellular switching mechanism already known to produce phenotypic alterations with strong correlations to changes in queuosine levels.

A comparative study of two "abnormal" human adenocarcinoma cell-lines (colon and breast) was performed to assess their queuosine levels and determine the malfunctioning system step(s) for the cause of the observed deficiencies. The 100% queuosine-deficient colon tumor cell-line possessed a null mutation for the queuosine incorporation enzyme, while the 50-60% queuosine-deficient breast tumor cell-line exhibited a strong deficiency in the queuine salvage mechanism. These results demonstrate the potential for determination of even multiple sites of lesions in the modification system that would yield queuosine-deficient tRNA characteristic of tumors.

Computational modeling was utilized to determine the biological function for the queuosine modification. Steric, electrostatic, and structural differences were observed for queuosine, queuosine-analogues and guanosine, the nucleosides incorporated into tRNAasp anticodon stem/loop structures, and in triad complexes of tRNAasp with mRNA and tRNAphe. The results of this research identify indistinguishable energetic parameters for complexes of queuosine-modified anticodon loops when paired with an mRNA containing cytosine- or a uridine-ending codon. However, guanosine-containing anticodon loops demonstrate much stronger energetic stability with cytosine-ending codons. The difference in codon bias is shown to be due to the restriction of anticodon loop flexibility by a queuosine-induced extended intraloop hydrogen bonding network and only minimally due to a shift in hydrogen bonding pattern produced by an intraresidue hydrogen bond.

A key difference in the physiology of normal and neoplastic cells is in the increased expression of oncodevelopmental genes with respect to those housekeeping genes needed for survival. Sequence analysis of several oncodevelopmental and housekeeping transcripts suggests the presence of a contrasting bias in the usage of queuosine-related codons which end in cytosine or either cytosine or uridine, respectively. In combination with the mechanism proposed for tRNAasp decoding preferences, this codon usage bias suggests a potentially influential role for the queuosine modification system in the translational control of oncodevelopmental gene expression.

Comments

Dissertation submitted to the Faculty of Eastern Virginia Medical School and Old Dominion University in Partial Fulfillment of the Requirement for the Degree of Doctor of Philosophy in Biomedical Sciences.

DOI

10.25777/bwhj-g976

ISBN

9780591603910

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