The ribosomal neighbourhood of the central fold of tRNA: cross-links from position 47 of tRNA located at the A, P or E site.
The naturally occurring nucleotide 3-(3-amino-3-carboxy-propyl)uridine (acp3U) at position 47 of tRNA(Phe) from Escherichia coli was modified with a diazirine derivative and bound to ribosomes in the presence of suitable mRNA analogues under conditions specific for the ribosomal A, P or E sites. After photo-activation at 350 nm the cross-links to ribosomal proteins and RNA were identified by our standard procedures. In the 30S subunit protein S19 (and weakly S9 and S13) was the target of cross-linking from tRNA at the A site, S7, S9 and S13 from the P site and S7 from the E site. Similarly, in the 50S subunit L16 and L27 were cross-linked from the A site, L1, L5, L16, L27 and L33 from the P site and L1 and L33 from the E site. Corresponding cross-links to rRNA were localized by RNase H digestion to the following areas: in 16S rRNA between positions 687 and 727 from the P and E sites, positions 1318 and 1350 (P site) and 1350 and 1387 (E site); in the 23S rRNA between positions 865 and 910 from the A site, 1845 and 1892 (P site), 1892 and 1945 (A site), 2282 and 2358 (P site), 2242 and 2461 (P and E sites), 2461 and 2488 (A site), 2488 and 2539 (all three sites) and 2572 and 2603 (A and P sites). In most (but not all) cases, more precise localizations of the cross-link sites could be made by primer extension analysis.
Max-Planck-Institut fuer Molekulare Genetik (AG Ribosomen), Berlin (Dahlem), Germany.
M
M
Osswald
T
T
Döring
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R
Brimacombe
D001483:Base Sequence; D001665:Binding Sites; D003432:Cross-Linking Reagents; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism / Q000648:ultrastructure; D008961:Models, Structural; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D012329:RNA, Bacterial / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D012333:RNA, Messenger / Q000737:chemistry / Q000378:metabolism; D012336:RNA, Ribosomal, 16S / Q000737:chemistry / Q000378:metabolism; D012343:RNA, Transfer / Q000737:chemistry*; D012358:RNA, Transfer, Met / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012360:RNA, Transfer, Phe / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism / Q000648:ultrastructure*
D003432:Cross-Linking Reagents; D012329:RNA, Bacterial; D012333:RNA, Messenger; D012336:RNA, Ribosomal, 16S; D012358:RNA, Transfer, Met; D012360:RNA, Transfer, Phe; C024988:tRNA, formylmethionine-; D012343:RNA, Transfer