Degree Type

Dissertation

Date of Award

1989

Degree Name

Doctor of Philosophy

Department

Biochemistry, Biophysics and Molecular Biology

First Advisor

Jack Horowitz

Abstract

The structural basis for the recognition of valine transfer RNA (tRNA[superscript] Val) by valyl-tRNA synthetase (VRS) from E. coli has been investigated by [superscript]19F NMR spectroscopy, aminoacylation kinetics and the technique or site-directed mutagenesis. The accomplishments presented here entail (1) the development of protocols for the synthesis of wild type and mutant tRNA[superscript] Val having 5-fluorouracil in place of uracil, (2) the direct assignment of [superscript]19F NMR peaks to specific FUra residues in tRNA[superscript] Val, and (3) the role played by specific bases in the recognition of tRNA[superscript] Val by its cognate synthetase;For in vitro synthesis of FUra-substituted tRNA[superscript] Val, plasmid pVAL119-21, combining a synthetic E. coli tRNA[superscript] Val gene, along with the T7 RNA promoter, was constructed. The synthetic tRNA gene has incorporated at its 3[superscript]' terminus the restriction site for BstN1. Runoff transcription by T7 RNA polymerase of the BstN1 digested plasmid yields tRNA[superscript] Val which has no minor bases. The tRNA transcript, purified by HPLC, has a valine acceptance activity of 1200-1300 pmole/A[subscript]260;The [superscript]19F NMR spectra of the transcribed and native (FUra)tRNA[superscript] Val are identical except for slight differences in the chemical shifts of resonances in the central region of the spectrum (peaks E and F). Comparison of [superscript]19F NMR spectra of wild type and mutant forms of (FUra)tRNA[superscript] Val leads to the assignment of peaks B, C, E, F, H, K and L to fluorouracil 64, 59, 47, 33, 34, 29 and 67 respectively;Aminoacylation kinetic studies show that most of the mutant tRNAs could be aminoacylated by VRS. However, replacement of C36 with A, G, or U results in 150-1000 fold decrease in V[subscript] max/K[subscript] m. Mutants C34 and G41 also have lower V[subscript] max/K[subscript] m values. Addition of VRS induces several changes in the [superscript]19F NMR spectrum of (FUra) tRNA[superscript] Val. Intensity at 5 ppm increases, while the signal at 4 ppm (peak G/H) loses intensity. One peak shifts downfield from the cluster around 3 ppm. These results suggest an involvement of the anticodon in the recognition of tRNA[superscript] Val by VRS.

DOI

https://doi.org/10.31274/rtd-180813-11543

Publisher

Digital Repository @ Iowa State University, http://lib.dr.iastate.edu/

Copyright Owner

Wen-Chy Chu

Language

en

Proquest ID

AAI8920117

File Format

application/pdf

File Size

165 pages

Included in

Biochemistry Commons

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