Site-directed cross-linking of mRNA analogues to the Escherichia coli ribosome; identification of 30S ribosomal components that can be cross-linked to the mRNA at various points 5' with respect to the decoding site.
Three different mRNA analogues (28 to 34 nucleotides long) were prepared by T7 transcription from synthetic DNA templates. Each message contained the sequence ACC-GCG (coding for threonine and alanine, respectively), together with a single thio-U residue located at a variable position on the 5'-side of these coding triplets. A photo-reactive group was introduced by substitution of the thio-U with 4-azidophenacyl bromide. The messages were bound to E. coli 70S ribosomes in the presence of the appropriate tRNA-Thr or tRNA-Ala, and the azidophenyl group was photoactivated. Cross-linking was found to occur exclusively within the 30S subunit, with the 32P-label in the cross-linked mRNA being divided roughly equally between 30S ribosomal proteins and 16S RNA. Immunological analysis of the cross-linked proteins showed that, in the presence of either tRNA species, protein S7 was the primary target, whereas in the absence of tRNA only small amounts of protein S21 were cross-linked. The cross-link site to 16S RNA lay in all cases very close to its extreme 3'-terminus. These data indicate that the outgoing message leaves the cleft of the 30S subunit in a "northerly" direction.
Stade
K
K
Max-Planck Institut für Molekulare Genetik, Abteilung Wittmann, Berlin-Dahlem, FRG.
Rinke-Appel
J
J
Brimacombe
R
R
D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D007202:Indicators and Reagents; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000138:chemical synthesis / Q000302:isolation & purification / Q000378:metabolism*; D012269:Ribosomal Proteins / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism* / Q000648:ultrastructure; D013698:Templates, Genetic; D014158:Transcription, Genetic
D007202:Indicators and Reagents; D012333:RNA, Messenger; D012269:Ribosomal Proteins; C040469:ribosomal protein S30