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 00:00:00 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-
• Journal Information: Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States), Vol. 45:6; Conference: 76. annual meeting of the Federation of American Society for Experimental Biology, Washington, DC, USA, 8 Jun 1986
• Country of Publication: United States
• Language: English

Related Subjects

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