T4 诱导的 RNA 连接酶连接单链寡核糖核苷酸。

T4-induced RNA ligase joins single-stranded oligoribonucleotides.

作者信息

Walker G C, Uhlenbeck O C, Bedows E, Gumport R I

出版信息

Proc Natl Acad Sci U S A. 1975 Jan;72(1):122-6. doi: 10.1073/pnas.72.1.122.

DOI:10.1073/pnas.72.1.122

PMID:1090929

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC432253/

Abstract

RNA ligase isolated from Escherichia coli infected with bacteriophage T4 will catalyze the formation of an intermolecular 3' leads to 5' phosphodiester linkage between an oligoribonucleotide with a free 3'-hydroxyl and another oligoribonucleotide with a 5'-phosphate. Upon reaction with (Ap)5C, nearly quantitative conversion of the hexamer [5'-32P]p(Up)5U to the dodecamer (Ap)5C[3' leads to 5'-32P]p(Up)5U was observed. The product was identified by its mobility on RPC-5 column chromatography, its resistance to alkaline phosphatase, and the appearance of the expected radiolabeled products on hydrolysis with alkali, ribonuclease A, snake venom phosphodiesterase, and spleen phosphodiesterase. The coupling of other pairs of single-stranded oligoribonucleotides has also been demonstrated. The intermolecular joining reaction is probably mechanistically similar to the intramolecular cyclization activity previously reported for Tr RNA ligase. It is expected that this enzyme will be useful for the synthesis of RNA fragments of defined sequence.

摘要

从感染噬菌体T4的大肠杆菌中分离出的RNA连接酶，能催化在一个具有游离3'-羟基的寡核糖核苷酸与另一个具有5'-磷酸的寡核糖核苷酸之间形成分子间3'至5'磷酸二酯键。与(Ap)5C反应时，观察到六聚体[5'-32P]p(Up)5U几乎定量转化为十二聚体(Ap)5C[3'至5'-32P]p(Up)5U。通过其在RPC-5柱色谱上的迁移率、对碱性磷酸酶的抗性以及在用碱、核糖核酸酶A、蛇毒磷酸二酯酶和脾磷酸二酯酶水解时预期放射性标记产物的出现来鉴定该产物。其他单链寡核糖核苷酸对的偶联也已得到证实。分子间连接反应在机制上可能类似于先前报道的Tr RNA连接酶的分子内环化活性。预计这种酶将有助于合成特定序列的RNA片段。

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