title: Neighbourhood of the central fold of the tRNA molecule bound to the E. coli ribosome--affinity labeling studies with modified tRNAs carrying photoreactive probes attached to the dihydrouridine loop.
abstract: The neighbourhood of the dihydrouridine loop of tRNA molecule bound to E. coli ribosome has been studied by affinity labeling, using modified tRNAs carrying photoreactive azidonitrophenyl probes attached to the 3-(3-amino-3-carboxypropyl)-uridine located at position 20:1 of Lupin methionine elongator tRNA. The maximum distance between the pyrimidine ring and the azido group estimated for the two probes employed in this study is 10-11 A and 18-19 A, respectively. Cross-linking of the uncharged, modified tRNAs has been studied with poly(A, U, G) as a message, under conditions directing uncharged tRNAs preferentially to the ribosomal P-site. Modified tRNAs bind covalently to both ribosomal subunits with high yields upon irradiation of the respective non-covalent complexes. Proteins S7, L33 and L1 have been consistently found cross-linked to tRNAs modified with both probes, and S5 and L5 to tRNA modified with the longer probe. Surprisingly, an S5-tRNA cross-linking product is reproducibly found in a protein fraction prepared from the purified 50S subunit. Cross-linking to rRNAs is significant only for the longer probe and is stimulated 2-4 fold in the presence of poly(A,U,G). The cross-linking sites are located between nucleotides 1302 and 1398 in 16S rRNA and between nucleotides 2281 and 2358 in 23S rRNA.
authors.affiliation: Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznan.
authors.forename: J
authors.initials: J
authors.lastname: Podkowinski
authors.forename: P
authors.initials: P
authors.lastname: Gornicki
mesh_terms: D000345:Affinity Labels; D003432:Cross-Linking Reagents; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D005798:Genes, Bacterial; D009690:Nucleic Acid Conformation; D015347:RNA Probes; D012329:RNA, Bacterial / Q000235:genetics*; D012358:RNA, Transfer, Met / Q000737:chemistry*; D012269:Ribosomal Proteins / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism*; D014529:Uridine / Q000031:analogs & derivatives* / Q000378:metabolism
chemical_list: D000345:Affinity Labels; D003432:Cross-Linking Reagents; D015347:RNA Probes; D012329:RNA, Bacterial; D012358:RNA, Transfer, Met; D012269:Ribosomal Proteins; C010922:5,6-dihydrouridine; D014529:Uridine