Clustering of modified nucleotides at the functional center of bacterial ribosomal RNA.
An aryl trifluoromethyl diazirine photoreactive derivative was attached to the 2-thiocytidine residue at position 32 of tRNA(IArg) and this derivatized tRNA was bound to Escherichia coli 70S ribosomes. After irradiation at 350 nm the site of cross-linking to the 16S RNA was analyzed by our standard procedures and found to lie within the secondary structural element comprising bases 956-983; this region contains two modified nucleotides at positions 966 and 967. Similarly, an aryl azido photoreactive derivative was attached to the phenylalanine residue of Phe-tRNA(Phe), and the derivatized aminoacyl tRNA was bound to the ribosome either at the A- or the P-site. In both cases, after irradiation at 250 nm, the cross-link site was localized to position 2439 of the 23S RNA; in the secondary structure of the latter the neighboring nucleotide 2442 is base-paired to a modified nucleotide at position 2069. Taken together with other cross-linking data, these results now directly implicate a total of 27 out of the 29 modified nucleotides in E. coli 16S and 23S RNA as lying within or close to the functional center of the ribosome.
Brimacombe
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R
Max-Planck-Institut für Molekulare Genetik, Berlin, Dahlem, Germany.
Mitchell
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P
Osswald
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M
Stade
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K
Bochkariov
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D
D001389:Azirines / Q000737:chemistry; D001483:Base Sequence; D001665:Binding Sites; D003432:Cross-Linking Reagents; D003562:Cytidine / Q000031:analogs & derivatives / Q000737:chemistry; D004926:Escherichia coli / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D009711:Nucleotides / Q000737:chemistry*; D012336:RNA, Ribosomal, 16S / Q000737:chemistry* / Q000378:metabolism / Q000528:radiation effects; D012347:RNA, Transfer, Arg / Q000737:chemistry / Q000378:metabolism / Q000528:radiation effects; D012360:RNA, Transfer, Phe / Q000737:chemistry / Q000378:metabolism / Q000528:radiation effects; D012270:Ribosomes / Q000737:chemistry* / Q000378:metabolism / Q000528:radiation effects; D014466:Ultraviolet Rays
D001389:Azirines; D003432:Cross-Linking Reagents; D009711:Nucleotides; D012336:RNA, Ribosomal, 16S; D012347:RNA, Transfer, Arg; D012360:RNA, Transfer, Phe; C002908:2-thiocytidine; D003562:Cytidine