Photochemical cross-linking of tRNA/sup Phe/ modified at A/sub 76/ and A/sub 73/ to the Escherichia coli ribosome

Conference · Thu May 01 04:00:00 EDT 1986 · Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States)

OSTI ID:5269282

Wower, J; Aymie, M; Hixson, S S; Zimmermann, R A

(5'-/sup 32/P)-8-azidoadenosine 3',5'-bisphosphate ((5'-/sup 32/P)p(N/sub 3/)Ap) has been prepared using a simple two-step procedure: alkaline hydrolysis of 8-azidoadenosine 3',5'-cyclic monophosphate followed by labeling of the resulting 3'-mononucleotide with /sup 32/P at the 5' position using (..gamma..-/sup 32/P)ATP and T4 polynucleotide kinase. (5'-/sup 32/P)p(N/sub 3/)Ap has proven to be an excellent substrate for T4 RNA ligase. To study the environment of the 3' end of tRNA on bacterial ribosomes, nucleosides A/sub 76/ and A/sub 73/ in yeast tRNA/sup Phe/ were replaced with their 8-azido derivatives. This was achieved by stepwise removal of 3'-terminal nucleosides from the tRNA using the Whitfield procedure, incorporation of (5'-/sup 32/P)p(N/sub 3/)Ap into appropriately degraded tRNA with T4 RNA ligase, and restoration of the CCA/sub OH/ terminus with yeast nucleotidyl transferase. The modified tRNAs were bound to the A or P site of Escherichia coli ribosomes programmed with poly(U). UV irradiation produced covalent, zero-length cross-links between the tRNA and neighboring ribosomal components. The tRNA derivative containing (N/sub 3/)A/sub 73/ became attached exclusively to proteins of the 50S subunit whose identity is currently under investigation.

• Research Organization: Univ. of Massachusetts, Amherst
• OSTI ID: 5269282
• Report Number(s): CONF-8606151-
• Conference Information: Journal Name: Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States) Journal Volume: 45:6
• Country of Publication: United States
• Language: English

Related Subjects

560131* -- Radiation Effects on Microorganisms-- Basic Studies-- (-1987)
63 RADIATION, THERMAL, AND OTHER ENVIRON. POLLUTANT EFFECTS ON LIVING ORGS. AND BIOL. MAT.
ATP
BACTERIA
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
BIOLOGICAL EFFECTS
BIOLOGICAL RADIATION EFFECTS
CELL CONSTITUENTS
CHEMICAL REACTIONS
CHEMISTRY
CROSS-LINKING
DAYS LIVING RADIOISOTOPES
ELECTROMAGNETIC RADIATION
ENZYMES
ESCHERICHIA COLI
FUNGI
GENETIC EFFECTS
GENETIC RADIATION EFFECTS
ISOTOPE APPLICATIONS
ISOTOPES
LIGASES
LIGHT NUCLEI
MICROORGANISMS
NUCLEI
NUCLEIC ACIDS
NUCLEOSIDES
NUCLEOTIDES
ODD-ODD NUCLEI
ORGANIC COMPOUNDS
ORGANOIDS
PHOSPHORUS 32
PHOSPHORUS ISOTOPES
PHOSPHORUS-GROUP TRANSFERASES
PHOSPHOTRANSFERASES
PHOTOCHEMISTRY
PLANTS
POLYMERIZATION
RADIATION EFFECTS
RADIATIONS
RADIOISOTOPES
RIBOSIDES
RIBOSOMES
RNA
TRACER TECHNIQUES
TRANSFER RNA
TRANSFERASES
ULTRAVIOLET RADIATION
YEASTS