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.
Department of Biochemistry and Molecular Biology, University of Massachusetts, Amherst 01003.
J
J
Wower
S S
SS
Hixson
L A
LA
Sylvers
Y
Y
Xing
R A
RA
Zimmermann
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