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

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Abstract
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 tRNAPhe with little effect on the amino acid acceptor activity of the tRNA. In addition, we show that the modified tRNAPhe derivative can be photochemically cross‐linked to the Escherichia coli ribosome.