Item Details

title

Experimental and Computational Dynamics of an Aminoacyl-tRNA Synthetase System

author

Macnamara, Lindsay

abstract

The fidelity of protein synthesis is dependent upon correct recognition and aminoacylation of transfer RNA (tRNA) by their cognate aminoacyl-tRNA synthetases (AARSs). Methionine is the amino acid that acts as the start codon for every protein and can also be found within a protein message. Methionyl-tRNA synthetase (MetRS) is responsible for the correct recognition and aminoacylation of tRNAMet. The tRNAMet acceptor stem must undergo a conformational change via allosteric signaling to fit into the enzyme active site. However, little is known about the signaling network within MetRS and tRNAMet propagate this change. To understand the communication between MetRS and tRNAMet, we made tRNA variants with single nucleotide substitutions in conserved core nucleotide, and MetRS variants with changes in the connective polypeptide. MetRS contains a highly mobile connective polypeptide (CP) domain which contains a β-linker, which is unique to MetRS species and a zinc binding knuckle. The length of the β-linker was altered to determine how it is important for structuring the zinc binding knuckle. Eight prolines are found in the CP domain which were individually mutated to alanine. Circular dichroism, binding affinity, and steady-state kinetics were evaluated to determine how the CP domain variants affect the structure and function of the zinc binding knuckle. Molecular dynamics simulations of the tRNA core variants and proline variants were used to investigate changes in structure and dynamics. This project looks at this question from both the experimental and computational methods to determine how this communication is facilitated.

contributor

Alexander, Rebecca W (committee chair)

Muday, Gloria (committee member)

Hollis, Thomas (committee member)

Poole, Leslie (committee member)

DosSantos, Patricia (committee member)

date

2018-01-17T09:35:30Z (accessioned)

2020-01-16T09:30:20Z (available)

2017 (issued)

degree

Biochemistry and Molecular Biology (discipline)

embargo

2020-01-16 (terms)

identifier

http://hdl.handle.net/10339/89871 (uri)

language

en (iso)

publisher

Wake Forest University

type

Dissertation