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.

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RESUMO

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.

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