High resolution localization of the tRNA anticodon interaction site on the Escherichia coli 30 S ribosomal subunit.
A body of previous work has shown that when Escherichia coli tRNAVal1 is placed in the P site of E. coli ribosomes and irradiated, the 5'-anticodon base of this tRNA, 5-carboxymethoxyuridine, is cross-linked to C-1400 of the 16 S rRNA. By tagging the carboxyl group of the cross-linked tRNA residue with a 2,4-dinitrophenyl (DNP) group attached via a 9 A spacer, it has been possible to directly visualize this cross-linking site by immunoelectron microscopy. The DNP group was attached by addition of ethylenediamine to the carboxyl group, followed by condensation of the newly formed free amino group with the N-hydroxysuccinimide ester of N-2,4-dinitrophenyl-gamma-aminobutyric acid. When reacted with anti-DNP antibody, this modification brings the surface of the antibody to within 9 A of the pyrimidine ring which was cross-linked. Neither codon-dependent binding nor cross-linking were materially affected by the tRNA modification. The tRNA-ribosome adduct formed a stable complex with anti-DNP antibody only when 50-30 S subunit association was prevented. Electron microscopic examination of the immune complexes showed that greater than 95% of those detected had the antibody localized deep in the cleft which separates the head and neck of the 30 S from the large protrusion. Since this is the site of cross-linking of the anticodon of tRNA, we conclude that this region on the 30 S subunit corresponds to the decoding site.
D000906:Antibodies; D000926:Anticodon / Q000378:metabolism*; D000936:Antigen-Antibody Complex; D001483:Base Sequence; D004926:Escherichia coli / Q000378:metabolism*; D008854:Microscopy, Electron; D008961:Models, Structural; D009690:Nucleic Acid Conformation; D012343:RNA, Transfer / Q000378:metabolism*; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism*
D000906:Antibodies; D000926:Anticodon; D000936:Antigen-Antibody Complex; D012346:RNA, Transfer, Amino Acyl; D012343:RNA, Transfer