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|Title:||Substitution of the Anticodon Loop of Yeast Tyrosine Transfer Ribonucleic Acid (Rna)|
|Author(s):||Bare, Lance Alan|
|Department / Program:||Biochemistry|
|Degree Granting Institution:||University of Illinois at Urbana-Champaign|
|Abstract:||Procedures for replacing residues 33-35 in the anticodon loop of yeast tRNA('Tyr) with any desired oligonucleotide have been developed. Half-molecules of tRNA('Tyr) from partial digestions with ribonuclease A or T(,1) were paired to create a gap in the anticodon loop. An oligonucleotide was inserted into the gap using RNA ligase, polynucleotide kinase, and Pset 1 polynucleotide kinase. The rate of aminoacylation of anticodon loop substituted tRNA('TYR)s by yeast tyrosyl-tRNA synthetase was found to depend upon the sequence of the oligonucleotide inserted. This suggests that the nucleotides in the anticodon loop of yeast tRNA('Tyr) are required for optimal aminoacylation. In addition, tRNA('Tyr) modified to have a phenylalanine anticodon was shown to be misacylated by yeast phenylalanyl-tRNA synthetase at a rate at least 10 times faster than unmodified tRNA('Tyr). Substitution of the anticodon of yeast tRNA('Phe) with a tyrosine anticodon was shown to stimulate the level of misacylation by tyrosyl-tRNA synthetase. Thus, the anticodon is used by phenylalanyl-tRNA synthetase and by the tyrosyl-tRNA synthetase to distinguish between tRNAs.
The aminoacylation kinetics of nineteen different variants of yeast tRNA('Tyr )with nucleotide substitutions in positions 33-35 were determined. Substitution of the conserved uridine-33 does not alter the rate of aminoacylation. However, substitution of the anticodon positions 34 or 35 reduce Km from 2- to 10-fold and Vmax as much as 2-fold depending on the nucleotide inserted. The ochre and amber suppressor tRNA('Tyr)s both showed about a 7-fold reduction in Vmax/Km. Data from tRNA('Tyr) with different modified nucleotides at position 35 suggest that specific hydrogen bonds form between the synthetase and both the N1 and N3 hydrogens of (PSI)-35. The effect of simultaneous substitutions at position 34 and 35 can be predicted reasonably well by combining the effects of single substitutions. These data suggest that yeast tyrosyl-tRNA synthetase interacts with position 34 and 35 of the anticodon of tRNA('Tyr) and that suppressor efficiency may be mediated by the level of aminoacylation.
Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1985.
|Date Available in IDEALS:||2014-12-15|