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|Title:||Substitution of "universal" Uridine-33 in yeast phenylalanine transfer ribonucleic (Rna)|
|Author(s):||Wittenberg, Wayne Layton|
|Department / Program:||Biochemistry|
|Degree Granting Institution:||University of Illinois at Urbana-Champaign|
|Abstract:||Functional groups of "universal" uridine-33 in yeast tRNA('Phe) were altered by a synthetic procedure which replaces U-33 with any desired nucleotide and leaves all other nucleotides of the tRNA intact. The U-33 substituted tRNAs were prepared in an eight step protocol that begins with partial cleavage of tRNA('Phe) at U-33 by ribonuclease A to give two tRNA('Phe) half-molecules. U-33 was removed from the 5' half-molecule in three steps to give a 5' half-molecule 1-32 terminating at the 3'-end in a 3' hydroxyl. Replacement nucleotides were added to the 5' half-molecule 1-32 as nucleoside 3',5'-bisphosphates with RNA ligase. A position 33 substituted 5' half-molecule and the original 3' half-molecule from ribonuclease A digestion were isolated, annealed, and repaired in three steps to give a full-size tRNA('Phe) variant.
Eight substituted tRNAs containing uridine, pseudouridine, deoxyuridine, 2'-O-methyluridine, 3-methyluridine, cytidine, deoxycytidine, and purine riboside at position 33 were prepared. All seven tRNAs that were tested aminoacylated normally with yeast phenylalanyl-tRNA synthetase. Thus U-33 is not involved in the interaction between tRNA('Phe) and the synthetase. The activity of the tRNAs in protein synthesis was assayed in two ways. In one assay, rates of GTP hydrolysis and dipeptide formation were determined for single-turnover reactions of poly(U)-programmed 70S ribosomes with EF-Tu ternary complexes of seven of the Phe-tRNAs in a rapid mixing apparatus. In the other assay, dissociation constants were determined for the binding of seven of the tRNAs to poly(U)-programmed 30S ribosomes. U-33 was not essential for the activity of tRNA('Phe) in either assay. However, for optimally efficient GTP hydrolysis and dipeptide formation, a uridine at position 33 was necessary. The tRNAs containing a nucleotide 33 with an altered uracil, or altered ribose, or both showed reduced rates of GTP hydrolysis and increased amounts of GTP hydrolysis per peptide bond formed. Similarly, a uridine at position 33 was necessary for optimal binding of tRNA('Phe) to 30S ribosomes. Thus simple rearrangements of atoms of the uracil and ribose of U-33 affect the codon-anticodon interaction in translation.
Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1984.
|Date Available in IDEALS:||2014-12-15|