Probing tRNA binding sites on the Escherichia coli 30 S ribosomal subunit with photoreactive analogs of the anticodon arm.
Two analogs of the anticodon arm of yeast tRNAPhe (residues 28-43), in which G43 was replaced by the photoreactive nucleosides 2-azidoadenosine and 8-azidoadenosine, have been used to create 'zero-length' cross-links to ribosomal components at the peptidyl-tRNA binding site (P site) of 30 S subunits from the Escherichia coli ribosome. To prepare the analogs, 2-azidoadenosine and 8-azidoadenosine bisphosphates were first ligated to the 3' end of the anticodon-containing dodecanucleotide ACmUGmAAYA psi m5CUG from yeast tRNAPhe. The trinucleotide CAG was then joined to the 5' end of the resulting tridecanucleotide in a subsequent ligation. Both analogs bound to poly(U)-programmed 30 S subunits with affinities similar to that of the unmodified anticodon arm from yeast tRNAPhe. Irradiation of noncovalent complexes containing the photolabile analogs, poly(U) and 30 S ribosomal subunits with 300 nm light led to the covalent attachment of the anticodon arms to proteins S13 and S19. Further analysis revealed that S13 accounted for about 80%, and S19 for about 20%, of the cross-linked material. Labeling of these two proteins with 'zero-length' cross-linking probes provides useful information about the location and orientation of P site-bound tRNA on the ribosome and permits a test of recently proposed models of the three-dimensional structure of the 30 S subunit.
Wower
J
J
Department of Biochemistry, University of Massachusetts, Amherst 01003.
Malloy
T A
TA
Hixson
S S
SS
Zimmermann
R A
RA
D000241:Adenosine / Q000031:analogs & derivatives; D000345:Affinity Labels; D000926:Anticodon / Q000378:metabolism*; D001386:Azides; D001483:Base Sequence; D001665:Binding Sites; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D007700:Kinetics; D008961:Models, Structural; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D015345:Oligonucleotide Probes / Q000138:chemical synthesis*; D012360:RNA, Transfer, Phe / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism* / Q000528:radiation effects / Q000648:ultrastructure
D000345:Affinity Labels; D000926:Anticodon; D001386:Azides; D015345:Oligonucleotide Probes; D012360:RNA, Transfer, Phe; C012760:8-azidoadenosine; C022294:2-azidoadenosine; D000241:Adenosine