title: The path of mRNA through the Escherichia coli ribosome; site-directed cross-linking of mRNA analogues carrying a photo-reactive label at various points 3' to the decoding site.
abstract: mRNA analogues approximately 40 bases long were prepared by T7 transcription from synthetic DNA templates. Each message contained the sequence ACC-GCG (coding for threonine and alanine, respectively), together with a single thio-U residue located at a variable position on the 3'-side of these coding triplets. The thio-U residue was either substituted with 4-azidophenacyl bromide to introduce a photo-reactive group, or was left unsubstituted for direct UV cross-linking. After binding to Escherichia coli 70S ribosomes in the presence of tRNA-Thr or tRNA-Ala, the thio-U residue or azidophenyl group was photo-activated and the products of cross-linking (which was exclusively to the 30S subunit) were analysed. Immunological analysis of the cross-linked proteins showed that S5 and S3, together with S1, were the targets of cross-linking at positions close to the decoding site, with the cross-linking to S3 and S1 persisting at positions further away. Analysis of the 16S RNA showed cross-links to the region of bases 1390-1400 in all cases, but in one instance (with the reactive nucleotide 11 bases from the decoding site) simultaneous cross-linking was observed to the latter region and to position 532; these two RNA regions are far apart in current three-dimensional models of the 30S subunit.
authors.affiliation: Max-Planck-Institut für Molekulare Genetik, Abteilung Wittmann, Berlin, FRG.
authors.forename: J
authors.initials: J
authors.lastname: Rinke-Appel
authors.forename: N
authors.initials: N
authors.lastname: Jünke
authors.forename: K
authors.initials: K
authors.lastname: Stade
authors.forename: R
authors.initials: R
authors.lastname: Brimacombe
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D003432:Cross-Linking Reagents; D004269:DNA, Bacterial / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism*; D008969:Molecular Sequence Data; D010777:Photochemistry; D012329:RNA, Bacterial / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D012336:RNA, Ribosomal, 16S / Q000378:metabolism; D012344:RNA, Transfer, Ala / Q000378:metabolism; D012363:RNA, Transfer, Thr / Q000378:metabolism; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism*; D013698:Templates, Genetic
chemical_list: D003432:Cross-Linking Reagents; D004269:DNA, Bacterial; D012329:RNA, Bacterial; D012333:RNA, Messenger; D012336:RNA, Ribosomal, 16S; D012344:RNA, Transfer, Ala; D012363:RNA, Transfer, Thr; D012194:RNA-Directed DNA Polymerase