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Nucleic Acids Research, 1993, Vol. 21, No. 4 887-896
© 1993

MOLECULAR BIOLOGY

Site-directed cross-linking studies on the E.coli tRNA ribosome complex: determination of sites labelled with an aromatic azide attached to the variable loop or aminoacyl group of tRNA

Philip Mitchell, Katrin Stade, Monika Oßwald and Richard Brimacombe*

Max-Planck-Instftut für Molekulare Genetik Abteilung Wittmann, Ihnestraße 73, 1000-Berlin 33, Germany

* To whom correspondence should be addressed

Received November 19, 1992. Revised December 21, 1992. Accepted December 21, 1992.

tRNAphe from E.coli, modified with the photoreactive label N-(p-azidobenzoyl)-glycine (ABG) either at the naturally occurring nucleotide 3-(3-amino-3-carboxypropyl) uridine (acp3U47 or the {alpha}-amino group of Phe-tRNAPhe, was bound nonenzymatically to 70S ribosomes in the presence of poly (U) or short synthetic mRNA molecules prepared by T7 transcription. The noncovalent complexes were subjected to a mild ultraviolet irradiation treatment and the sites of photo-incorporation were analysed. When the photo-affinity label was attached to the aminoacyl group cross-linking was observed from both A- and P-site bound tRNA and involved exclusively the 50S subunit. In both cases the major target of cross-linking was a single site in 23S RNA, localized to position A-2439. A lower yield of cross-linking to L27 from both P- and A-sites was also observed. In contrast, cross-linking from the acp3U47 derivative was specific for P-site bound tRNA and involved mainly (but not exclusively) the 50S subunit. In this case rRNA and ribsomal protein were labelled in approximately equal yields, the sites of cross-linking involving A-2309 in 23S RNA and L33. These results are discussed in the light of our present knowledge concerning the structural arrangement of the tRNA-ribosome complex.


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