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

(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.