Potential sites of the complementary interaction of the translation initiation region (TIR) with 16S rRNA are revealed, and the role of these sites in the gene expression level is studied. The high expression level of a gene depends not only on the complementary interaction of TIR with 16S rRNA in sites proximal to the start codon [anti-Shine-Dalgarno (ASD) (delta G -8 to -10 kcal/mol) and downstream box (DB)] and located at the -15 to +20 mRNA region but also on complementary interactions in distal sites of the untranslated branch of TIR (mTIR). Among them, the UB (upsteam box) 1 site, complementarily interacting with the exposed 452-490 segment of the 440-490 loop of 16S rRNA, may be located in the -15 to -50 mTIR segment. In the -50 to -70 mTIR segment may be located UB2 and UB3 sites, which interact with the exposed segment 478-488 of the 440-490 loop and segment 520-532 of the 520-540 loop of 16S rRNA, the UB3 site being much more efficacious. The high expression level requires that the total free energy of complementary interactions of UB1, UB2, and UB3 sites with 16S rRNA exceeds -20 kcal/mol.