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Publication Date

2008-09-22

Availability

UM campus only

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PHD)

Department

Biochemistry and Molecular Biology (Medicine)

Date of Defense

2008-08-27

First Committee Member

Antoni Barrientos

Second Committee Member

Rudolf Werner

Third Committee Member

Zafar Nawaz

Fourth Committee Member

Murray P. Deutscher

Fifth Committee Member

Micheal Ibba

Abstract

The aminoacyl-tRNA synthetases are among the major protein components in the translation machinery. These essential proteins are responsible for charging their cognate tRNAs with the correct amino acid. Mammalian arginyl-tRNA synthetase (ArgRS), unlike all other eukaryotic aminoacyl-tRNA synthetases, is unique due to the coexistence of two structurally distinct forms of the same enzyme within the same cell: a complexed (or high molecular weight) form that is part of the multi-synthetase complex, and a free (or low molecular weight) form. Until now, not much information is known as to why the cell would synthesize and utilize two different forms of the same enzyme. Do the two forms of ArgRS perform similar or different biological functions? The main hypothesis that was originally proposed is that only the complexed form of ArgRS plays a crucial role in protein synthesis, while the free form of this enzyme participates in the ubiquitination pathway by tagging proteins with acidic NH2-termini (destined for degradation) with an arginine residue on their NH2-terminal end which will serve as a signal for ubiquitin-mediated destruction. Based on my studies, the data indicate that the high molecular weight form of ArgRS, which is present exclusively as an integral component of the multisynthetase complex, is essential for normal protein synthesis and growth of CHO cells even when low molecular weight, free ArgRS is present and Arg-tRNA continues to be synthesized at close to wild type levels. Based on these observations, we can conclude that Arg-tRNA generated by the synthetase complex is a more efficient precursor for protein synthesis than Arg-tRNA generated by free ArgRS, exactly as would be predicted by the channeling model for mammalian translation. No phenotype has been determined for cells expressing only the complexed form of ArgRS, and no direct interaction has been observed between ArgRS and arginyl-tRNA-protein transferase (ATE). Based on this information, we suggest that the function(s) of the free form of ArgRS is either not necessary or is performed by the complexed form when the free form is missing.

Keywords

Multienzyme Complexes; Protein Synthesis; TRNA Channeling; Aminoacylation

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