Biomedical subjects
A G Bruce
Publications and source records attributed to A G Bruce.
Replacement of anticodon loop nucleotides to produce functional tRNAs: amber suppressors derived from yeast tRNAPhe.
The method of anticodon loop replacement has been used to make derivatives of yeast tRNAPhe. By constructing tRNAs with a CUA anticodon, complementary to the amber (UAG) terminator, functional amber suppressor tRNAs were produced. The activity of these tRNAs was assayed in a mammalian cell-free protein synthesizing system. The level of suppression reflects the efficiency of codon recognition. tRNAs were constructed with either A, C, U, or G on the 3' side of the CUA anticodon. The tRNAs containing the purines were efficient amber suppressors, whereas those containing pyrimidines were inefficient.
Specific labeling of 3' termini of RNA with T4 RNA ligase.
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Reactions at the termini of tRNA with T4 RNA ligase.
T4 RNA ligase will catalyze the addition of nucleoside 3', 5'-bisphosphates onto the 3' terminus of tRNA resulting in tRNA molecule one nucleotide longer with a 3' terminal phosphate. Under appropriate conditions the reaction is quantitative and, if high specific radioactivity bisphosphates are used, it provides an efficient means for in vitro labeling of tRNA. Although the 3' terminal hydroxyl is a good acceptor, the 5' terminal phosphate in most tRNA's is not an effective donor in the RNA ligase reaction. This poor reactivity is due to the secondary structure of the 5' terminal nucleotide. If E. Coli tRNAf Met is used, the 5' phosphate is reactive and the major product with RNA ligase is the cyclic tRNA.
A summer school for parents of autistic children.
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Uridine-33 in yeast tRNA not essential for amber suppression.
The nucleotide at position 33 on the 5' side of the anticodon of almost all tRNAs is a uridine. Crystallographic studies of different tRNAs reveal that although the precise orientation of uridine-33 is not always the same, it connects the anticodon stacked along the 3' side of the loop with the pyrimidine-32 stacked on the 5' side of the loop. The remarkably conserved nature of uridine-33 and its unique position in the anticodon loop structure has led to suggestions that this nucleotide has an essential role in the translational mechanism. We have developed a biochemical procedure to replace nucleotides 33-35 in yeast tRNATyr with any desired sequence and used it to construct amber suppressor tRNAs having different nucleotides at position 33. As all of these synthetic amber suppressor tRNAs functioned well in eukaryotic in vitro suppression assays, we conclude that uridine-33 does not have an obligatory role in the translation mechanism.