Synthesis of 2,6-diazido-9-(beta-D-ribofuranosyl)purine 3',5'-bisphosphate: incorporation into transfer RNA and photochemical labeling of Escherichia coli ribosomes.
2,6-Diazido-9-(beta-D-ribofuranosyl)purine was prepared by the reaction of 2,6-dichloro-9-(beta-D-ribofuranosyl)purine with sodium azide. The nucleoside was bisphosphorylated with pyrophosphoryl chloride to form 2,6-diazido-9-(beta-D-ribofuranosyl)purine 3',5'-bisphosphate. This product was labeled with 32P using T4 polynucleotide kinase to exchange the 5' phosphate with the gamma phosphate of [gamma-32P]ATP. When yeast tRNA(Phe) containing 2,6-diazido-9-(beta-D-ribofuranosyl)purine at the 3' terminus was bound to the P site of the Escherichia coli ribosome in the presence of poly(U) and irradiated with 300-nm light, the photoreactive tRNA derivative became cross-linked exclusively to the 50S subunit. The label was attached to proteins L27 and L33 as well as to the 23S rRNA.
Wower
J
J
Department of Biochemistry and Molecular Biology, University of Massachusetts, Amherst 01003.
Hixson
S S
SS
Sylvers
L A
LA
Xing
Y
Y
Zimmermann
R A
RA
D000241:Adenosine / Q000031:analogs & derivatives* / Q000138:chemical synthesis; D000345:Affinity Labels / Q000138:chemical synthesis*; D001386:Azides / Q000138:chemical synthesis*; D002499:Centrifugation, Density Gradient; D003432:Cross-Linking Reagents / Q000138:chemical synthesis*; D004926:Escherichia coli / Q000737:chemistry* / Q000648:ultrastructure; D006863:Hydrogen-Ion Concentration; D008968:Molecular Conformation; D010777:Photochemistry; D012331:RNA, Fungal / Q000737:chemistry; D012343:RNA, Transfer / Q000737:chemistry*; D012360:RNA, Transfer, Phe / Q000737:chemistry; D012270:Ribosomes / Q000737:chemistry*; D013056:Spectrophotometry, Ultraviolet; D013395:Sucrose; D015003:Yeasts / Q000378:metabolism
C088352:2,6-diazido-9-(ribofuranosyl)purine 3',5'-bisphosphate; D000345:Affinity Labels; D001386:Azides; D003432:Cross-Linking Reagents; D012331:RNA, Fungal; D012360:RNA, Transfer, Phe; D013395:Sucrose; D012343:RNA, Transfer; D000241:Adenosine