Synthesis of a chemically reactive analog of the initiation codon: its reaction with ribosomes of Escherichia coli.
Nitrophenylated 5'-adenylic acid could be employed as primer in a polyribonucleotide nucleotidyltransferase (Micrococcus luteus) reaction to yield 5'-nitrophenylated pA-U-G. After reduction and subsequent bromoacetylation, an A-U-G analog was obtained, which could be used as an affinity label for the ribosomal A-U-G-binding site(s). After incubating the A-U-G affinity label with 70S ribosomes, 30S subunits programmed for initiation-factor-dependent fMet-tRNAMetf binding were obtained. Hence, the A-U-G analog had irreversibly reacted at the ribosomal decoding site. Initiation complexes which were formed with the labeled 30S subunits were puromycin-resistant. Furthermore, GTP hydrolysis, necessary for proper accommodation of initiator tRNA at the ribosomal donorsite, did not function in these complexes. These data indicate that immobilization of A-U-G at the decoding site of the ribosome allows factor-dependent initiator tRNA binding, but impairs accommodation at the donor site. The ribosomal protein(s) to which A-U-G was covalently bound at the decoding site were identified by polyacrylamide gel electrophoresis in the presence of urea or sarkosyl. The predominant affinity-labeled protein was found to be protein S18. Variation of the incubation conditions of the affinity-labeling reaction leads to attachment of A-U-G label to another ribosomal protein, S4, the ram gene product.
D000227:Adenine Nucleotides / Q000378:metabolism; D000345:Affinity Labels; D001426:Bacterial Proteins / Q000378:metabolism; D002499:Centrifugation, Density Gradient; D003062:Codon; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism*; D006150:Guanine Nucleotides / Q000378:metabolism; D007700:Kinetics; D008837:Micrococcus / Q000201:enzymology; D009596:Nitrophenols; D009713:Nucleotidyltransferases / Q000378:metabolism; D010448:Peptide Initiation Factors; D011119:Polynucleotides / Q000378:metabolism; D011485:Protein Binding; D012333:RNA, Messenger; D012343:RNA, Transfer / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism*; D014500:Uracil Nucleotides / Q000378:metabolism
D000227:Adenine Nucleotides; D000345:Affinity Labels; D001426:Bacterial Proteins; D003062:Codon; D006150:Guanine Nucleotides; D009596:Nitrophenols; D010448:Peptide Initiation Factors; D011119:Polynucleotides; D012333:RNA, Messenger; D014500:Uracil Nucleotides; D012343:RNA, Transfer; D009713:Nucleotidyltransferases