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.
tRNA(Phe) from E. coli, modified with the photoreactive label N-(p-azidobenzoyl)-glycine (ABG) either at the naturally occurring nucleotide 3-(3-amino-3-carboxy-propyl) uridine (acp3U47) or the alpha-amino group of Phe-tRNA(Phe), 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 ribosomal 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.
Mitchell
P
P
Max-Planck-Institut für Molekulare Genetik, Abteilung Wittmann, Berlin, Germany.
Stade
K
K
Osswald
M
M
Brimacombe
R
R
D000345:Affinity Labels; D001386:Azides; D001483:Base Sequence; D001665:Binding Sites; D003432:Cross-Linking Reagents; D004926:Escherichia coli / Q000235:genetics*; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010777:Photochemistry; D012329:RNA, Bacterial / Q000378:metabolism*; D012335:RNA, Ribosomal / Q000378:metabolism; D012360:RNA, Transfer, Phe / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism*; D014466:Ultraviolet Rays
D000345:Affinity Labels; D001386:Azides; D003432:Cross-Linking Reagents; D012329:RNA, Bacterial; D012335:RNA, Ribosomal; D012360:RNA, Transfer, Phe; C046991:N-hydroxysuccinimidyl-4-azidobenzoyl glycine