The decoding region of 16S RNA; a cross-linking study of the ribosomal A, P and E sites using tRNA derivatized at position 32 in the anticodon loop.
A photo-reactive diazirine derivative was attached to the 2-thiocytidine residue at position 32 of tRNA(Arg)I from Escherichia coli. This modified tRNA was bound under suitable conditions to the A, P or E site of E.coli ribosomes. After photo-activation of the diazirine label, the sites of cross-linking to 16S rRNA were identified by our standard procedures. Each of the three tRNA binding sites showed a characteristic pattern of cross-linking. From tRNA at the A site, a major cross-link was observed to position 1378 of the 16S RNA, and a minor one to position 936. From the P site, there were major cross-links to positions 693 and to 957 and/or 966, as well as a minor cross-link to position 1338. The E site bound tRNA showed major cross-links to position 693 (identical to that from the P site) and to positions 1376/1378 (similar, but not identical, to the cross-link observed from the A site). Immunological analysis of the concomitantly cross-linked ribosomal proteins indicated that S7 was the major target of cross-linking from all three tRNA sites, with S11 as a minor product. The results are discussed in terms of the overall topography of the decoding region of the 30S ribosomal subunit.
Döring
T
T
Max-Planck-Institut für Molekulare Genetik, Berlin, Germany.
Mitchell
P
P
Osswald
M
M
Bochkariov
D
D
Brimacombe
R
R
D000926:Anticodon / Q000737:chemistry / Q000378:metabolism; D001389:Azirines; D001483:Base Sequence; D001665:Binding Sites; D003432:Cross-Linking Reagents; D003562:Cytidine / Q000031:analogs & derivatives / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010441:Peptide Chain Elongation, Translational / Q000235:genetics; D012329:RNA, Bacterial / Q000737:chemistry / Q000378:metabolism; D012333:RNA, Messenger / Q000138:chemical synthesis / Q000378:metabolism; D012336:RNA, Ribosomal, 16S / Q000378:metabolism*; D012347:RNA, Transfer, Arg / Q000737:chemistry / Q000378:metabolism*; D016914:Ribonuclease H; D012270:Ribosomes / Q000378:metabolism; D046249:Transfer RNA Aminoacylation; D014466:Ultraviolet Rays
D000926:Anticodon; D001389:Azirines; D003432:Cross-Linking Reagents; D012329:RNA, Bacterial; D012333:RNA, Messenger; D012336:RNA, Ribosomal, 16S; D012347:RNA, Transfer, Arg; C002908:2-thiocytidine; D003562:Cytidine; D016914:Ribonuclease H