Preparation of 2‐azidoadenosine 3′,5′‐[5′‐32P]bisphosphate for incorporation into transfer RNA Photoaffinity labeling of Escherichia coli ribosomes

FEBS Letters

Hixson, Stephen S.²  Zimmermann, Robert A.¹  Sylvers, Lee A.¹  Wower, Jacek¹ 

[1]Department of Biochemistry, University of Massachusetts, Amherst, MA 01003, USA

[2]Department of Chemistry, University of Massachusetts, Amherst, MA 01003, USA

关键词: Photoreactive nucleotide;    Polynucleotide kinase;    RNA ligase;    tRNA;    Photoaffinity labeling;    Ribosome;    2N3A;    2-azidoadenosine;    8N3A;    8-azidoadenosine;    2N3AMP;    2-azidoadenosine 5′-monophosphate;    pAp;    adenosine 2′;    5′- and 3′;    5′-bisphosphates (mixed isomers);    p2N3Ap;    2-azidoadenosine 2′;    5′- and 3′;    5′-bisphosphates (mixed isomers);    p8N3Ap;    8-azidoadenosine 2′;    5′- and 3′;    5′-bisphosphates (mixed isomers);    AcPhe-;    N-acetylphenylalanyl-;    PNK;    T4 polynucleotide kinase;    PEI;    polyethyleneimine;   

DOI  :  10.1016/0014-5793(89)80180-2

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

2-Azidoadenosine was synthesized from 2-chloroadenosine by sequential reaction with hydrazine and nitrous acid and then bisphosphorylated with pyrophosphoryl chloride to form 2-azidoadenosine 3′,5′-bisphosphate. The bisphosphate was labeled in the 5′-position using the exchange reaction catalyzed by T₄ polynucleotide kinase in the presence of [γ-³²P]ATP. Polynucleotide kinase from a T₄ mutant which lacks 3′-phosphatase activity (ATP:5′-dephosphopolynucleotide 5′-phosphotransferase, EC 2.7.1.78) was required to facilitate this reaction. 2-Azidoadenosine 3′,5′-[5′-³²P]bisphosphate can serve as an efficient donor in the T₄ RNA ligase reaction and can replace the 3′-terminal adenosine of yeast tRNA^Phe with little effect on the amino acid acceptor activity of the tRNA. In addition, we show that the modified tRNA^Phe derivative can be photochemically cross-linked to the Escherichia coli ribosome.

【 授权许可】

Unknown