From stand-by to decoding site. Adjustment of the mRNA on the 30S ribosomal subunit under the influence of the initiation factors.

The hypothesis of an adjustment of the mRNA in its ribosomal channel under the influence of the initiation factors has been tested by site-directed crosslinking experiments. Complexes containing 30S subunits with bound mRNA having 4-thio-uracil at specific positions were prepared in the presence or absence of initiation factors and/or fMet-tRNA and subjected to UV irradiation to obtain specific crosslinks of the radioactively labeled mRNA with bases of the 16S rRNA and with ribosomal proteins. The subsequent identification of the specific sites of both mRNA and rRNA and individual ribosomal proteins involved in the crosslinking, obtained under different conditions of complex formation, provide direct evidence for the occurrence of a partial relocation of the mRNA on the 30S ribosomal subunits under the influence of the factors. The nature of this mRNA relocation is compatible with our previous proposal of a shift of the template from an initial ribosomal "stand-by site" to a second site closer to that occupied when the initiation triplet of the mRNA is decoded in the P-site.