Labeling the peptidyltransferase center of the Escherichia coli ribosome with photoreactive tRNA(Phe) derivatives containing azidoadenosine at the 3' end of the acceptor arm: a model of the tRNA-ribosome complex.
Photoreactive derivatives of yeast tRNA(Phe) containing 2-azidoadenosine (2N3A) at position 73 or 76 have been crosslinked to the peptidyl site of Escherichia coli ribosomes. Covalent tRNA-ribosome attachment was dependent upon the replacement of adenosine by 2N3A in the tRNA, irradiation with 300-nm light, and the presence of poly(U). In all cases, the modified tRNAs became crosslinked exclusively to 50S ribosomal subunits. While the tRNA derivative containing 2N3A at position 73 labeled only protein L27, that containing 2N3A at position 76 labeled proteins L15, L16, and L27 as well as a segment of the 23S rRNA. The site of crosslinking in the rRNA was identified as guanosine-1945, which lies within a highly conserved sequence adjacent to a number of modified bases and has not until now been identified at the peptidyltransferase center. On the basis of these results, and previously reported crosslinks from tRNA containing 8-azidoadenosine in the 3'-terminal -A-C-C-A sequence [Wower, J., Hixson, S. S. & Zimmermann, R. A. (1988) Biochemistry 27, 8114-8121], we propose a model for the arrangement of tRNA molecules at the peptidyl and aminoacyl sites that is consistent with most of the information available about the location of the peptidyltransferase center and the decoding domain of the E. coli ribosome.
Wower
J
J
Department of Biochemistry, University of Massachusetts, Amherst 01003.
Hixson
S S
SS
Zimmermann
R A
RA
D000217:Acyltransferases / Q000378:metabolism*; D000241:Adenosine / Q000031:analogs & derivatives* / Q000494:pharmacology; D001386:Azides / Q000494:pharmacology*; D003432:Cross-Linking Reagents / Q000494:pharmacology*; D004926:Escherichia coli / Q000201:enzymology*; D008961:Models, Structural; D010458:Peptidyl Transferases / Q000378:metabolism*; D012338:RNA, Ribosomal, 23S / Q000378:metabolism; D012343:RNA, Transfer / Q000378:metabolism; D012345:RNA, Transfer, Amino Acid-Specific / Q000378:metabolism*; D012360:RNA, Transfer, Phe / Q000378:metabolism*; D012269:Ribosomal Proteins / Q000302:isolation & purification / Q000378:metabolism*; D012270:Ribosomes / Q000201:enzymology* / Q000648:ultrastructure; D013329:Structure-Activity Relationship
D001386:Azides; D003432:Cross-Linking Reagents; D012338:RNA, Ribosomal, 23S; D012345:RNA, Transfer, Amino Acid-Specific; D012360:RNA, Transfer, Phe; D012269:Ribosomal Proteins; C022294:2-azidoadenosine; D012343:RNA, Transfer; D000217:Acyltransferases; D010458:Peptidyl Transferases; D000241:Adenosine