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Preparation of 2‐azidoadenosine 3′,5′‐[5′‐ 32 P]bisphosphate for incorporation into transfer RNA Photoaffinity labeling of Escherichia coli ribosomes
Author(s) -
Sylvers Lee A.,
Wower Jacek,
Hixson Stephen S.,
Zimmermann Robert A.
Publication year - 1989
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(89)80180-2
Subject(s) - chemistry , rna ligase , escherichia coli , transfer rna , phosphotransferase , biochemistry , ribosome , rna , enzyme , dna ligase , gene
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 T 4 polynucleotide kinase in the presence of [γ‐ 32 P]ATP. Polynucleotide kinase from a T 4 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′‐ 32 P]bisphosphate can serve as an efficient donor in the T 4 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.