The path of mRNA through the Escherichia coli ribosome; site-directed cross-linking of mRNA analogues carrying a photo-reactive label at various points 3' to the decoding site

Abstract

mRNA analogues approximately 40 bases 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 3'-side of these coding triplets. The thio-U residue was either substituted with 4-azidophenacyl bromide to introduce a photo-reactive group, or was left unsubstituted for direct UV cross-linking. After binding to Escherichia coli 70S ribosomes in the presence of tRNA-Thr or tRNA-Ala, the thio-U residue or azidophenyl group was photo-activated and the products of cross-linking (which was exclusively to the 30S subunit) were analysed. Immunological analysis of the cross-linked proteins showed that S5 and S3, together with S1, were the targets of cross-linking at positions close to the decoding site, with the cross-linking to S3 and S1 persisting at positions further away. Analysis of the 16S RNA showed cross-links to the region of bases 1390-1400 in all cases, but in one instance (with the reactive nucleotide 11 bases from the decoding site) simultaneous cross-linking was observed to the latter region and to position 532; these two RNA regions are far apart in current three-dimensional models of the 30S subunit.