Preparation of 2-azidoadenosine 3',5'-[5'-32P]bisphosphate for incorporation into transfer RNA. Photoaffinity labeling of Escherichia coli ribosomes.
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 [gamma-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.
Sylvers
L A
LA
Department of Biochemistry, University of Massachusetts, Amherst 01003.
Wower
J
J
Hixson
S S
SS
Zimmermann
R A
RA
D000244:Adenosine Diphosphate / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000378:metabolism; D000345:Affinity Labels; D001386:Azides; D002855:Chromatography, Thin Layer; D004926:Escherichia coli / Q000378:metabolism*; D007700:Kinetics; D010766:Phosphorylation; D010777:Photochemistry; D011116:Polynucleotide 5'-Hydroxyl-Kinase / Q000378:metabolism; D012343:RNA, Transfer / Q000378:metabolism*; D012360:RNA, Transfer, Phe / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism*; D013604:T-Phages / Q000201:enzymology
D000345:Affinity Labels; D001386:Azides; D012360:RNA, Transfer, Phe; D000244:Adenosine Diphosphate; C022295:2-azidoadenosine 3',5'-diphosphate; D012343:RNA, Transfer; D011116:Polynucleotide 5'-Hydroxyl-Kinase