Nucleotides in yeast phenylanyl - transfer-RNA required for the specific recognition by its cognate yeast phenylalanyl - transfer-RNA synthetase
Sampson, Jeffrey Robert
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https://hdl.handle.net/2142/19990
Description
Title
Nucleotides in yeast phenylanyl - transfer-RNA required for the specific recognition by its cognate yeast phenylalanyl - transfer-RNA synthetase
Author(s)
Sampson, Jeffrey Robert
Issue Date
1989
Doctoral Committee Chair(s)
Clark, John M., Jr.
Department of Study
Biochemistry
Discipline
Biochemistry
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Chemistry, Biochemistry
Language
eng
Abstract
A method for synthesizing yeast tRNA$\sp{\rm Phe}$ mutants having nucleotide substitutions at any desired position in the tRNA has been developed. Using synthetic DNA oligonucleotides, recombinant plasmids were constructed with a T7 promoter directly upstream of the tRNA gene and a BstN 1 restriction site at the 3$\sp\prime$ terminus of the gene. Run-off transcription of the wild type plasmid gave a 76 nucleotide RNA having the sequence of yeast tRNA$\sp{\rm Phe}$ but lacking the 14 modified nucleosides normally present in yeast tRNA$\sp{\rm Phe}$. This unmodified tRNA$\sp{\rm Phe}$ transcript is aminoacylated by yeast phenylalanyl-tRNA synthetase (FRS) with similar kinetics to that of the fully modified yeast tRNA$\sp{\rm Phe}$. Using this method, 35 tRNA$\sp{\rm Phe}$ mutants were synthesized and their aminoacylation kinetics with FRS determined. In every case where the substitutions of the tertiary nucelotides resulted in a mutant which should maintain the tertiary structure of the tRNA, little effect on the aminoacylation kinetics was observed. However, nucleotide substitutions which disrupted a given tertiary interaction generally resulted in a lowered rate of aminoacylation. This suggests that these nucleotides are not involved in specific interactions with the synthetase but are required to maintain the proper folding of the tRNA necessary for the recognition by FRS. In contrast, substitution of the single stranded nucelotides G20, G34, A35, A36 and A73 resulted in large decreases in the rate of aminoacylation. When E. coli tRNA$\sp{\rm Phe}$ and three other yeast tRNAs were subsequently changed to have these five single stranded nucleotides, they became virtually normal substrates for FRS. This suggests that G20, G34, A35, A36 and A73 are important recognition nucelotides for FRS.
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