Modification of Transfer Rna With T4 Rna Ligase

Bruce, Allen Gregory

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https://hdl.handle.net/2142/67412 Copy

Description

Title

Modification of Transfer Rna With T4 Rna Ligase

Author(s)

Bruce, Allen Gregory

Issue Date

1981

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

• T4 RNA ligase can be used to alter the sequence of transfer RNAs. This enzyme will catalyze the addition of a 3',5'-bisphosphate onto the 3' terminus of a tRNA resulting in a tRNA molecule one nucleotide longer with a 3' 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 tRNAs is not an effective donor in the T4 RNA ligase reaction. This poor reactivity is due to the secondary structure at the 5' terminus. If E. coli tRNA('fMet) is used, the 5' phosphate is reactive and the major product with T4 RNA ligase is the cyclic tRNA.
• T4 RNA ligase can also be used to replace nucleotides in the anticodon loop of yeast tRNA('Phe). A tetranucleotide which includes the anticodon and the hypermodified nucleotide, G(,m)AAY, can be specifically removed by a series of chemical and enzymatic reactions. T4 RNA ligase and polynucleotide kinase can then be used to insert a new oligoribonucleotide. By inserting a series of thirteen different oligoribonucleotides whose sequences differ by a single nucleotide and determining the affects of these changes on the aminoacylation reaction, the wobble base, G-34, has been shown to be involved in the recognition of this tRNA by yeast phenylalanyl-tRNA synthetase.

Graduation Semester

1981

Type of Resource

text

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http://hdl.handle.net/2142/67412

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