## 3 Results
Error bars show standard deviations. landing region and thus physically blocks translation initiation. However, given that the H\({}_{1}\) hairpin is located in a region that is conserved in the _ilvBNC_ operon in _Corynebacterium_[6], the role of this hairpin remains unclear and may not be related to an attenuation mechanism as in some bacterial species in which the expression of the _ilvBNC_ operon is controlled by a ppGpp-sensitive riboswitch mechanism [7]. Given the above-mentioned data and the fact that the enzymatic probing of the secondary structure of the region of interest in _C. glutamicum_ was performed on another _Corynebacterium_ species, _C. ulcerans_, we compared the sequences in a narrower sample of _Corynebacterium_ species than that in [6], namely, in the 20 species in which the sequences of this region are closest to those of _C. glutamicum_, including _C. glutamicum_. Thus, the nucleotide positions in the regulatory region of the _ilvBNC_ operon that are conserved in the sample of _Corynebacterium_ species closest to _C. glutamicum_ were identified. However, as can be seen in Figure 3, where these positions are marked in gray, the conserved nucleotides at positions 36-64 are equally involved in both H\({}_{1}\) (AAT) and H\({}_{2}\) (AT) hairpin formation, making it impossible to confidently distinguish between them using evolutionary and enzymatic analyses. All three hairpins, AAT, AT, and T, have comparable stability (Figure 3), but the AT and AAT hairpins unfold as a result of interaction with the ribosome. Therefore, to a first approximation, changes in the gene transcription level caused by mutations in the attenuator will be proportional to the mutational perturbations of terminator stability. More specifically, to describe the mechanism of attenuation by a structural model (Figure 3), we compared the change in the transcription level of the _ilvBNC_ operon with the change in H\({}_{3}\) hairpin free energy as influenced by base pairing changes. The difference between the  
Figure 3: Thermodynamically stable mRNA secondary structures, the formation of which is possible in the regulatory region of the _ilvBNC_ operon in _C. glutamicum_. (**A**)—state of stopped transcription; (**B**)—intermediate structures responsible for the rapid transition between the terminator and antiterminator hairpins by the low-energy switching of nucleotides 76–78 from pairing with nucleotides 111–113 to pairing with nucleotides 36–38; (**C**)—state of transcription. Red arrows indicate the positions of single-nucleotide substitutions. Positions conserved in the attenuator sequences of the 19 _Corynebacterium_ species closest to _C. glutamicum_ are marked in gray. Denotations: T—terminator hairpin; AT—antiterminator hairpin; AAT—antiantiterminator hairpin. hairpin free energies was calculated on the basis of the thermodynamic parameters of the energies of noncomplementary pairs [19; 20] using the formula  
\[\Delta\Omega=\Delta G_{\mathrm{mT}}-\Delta G_{\mathrm{T}} \tag{1}\]  
where \(\Delta G_{\mathrm{mT}}\) and \(\Delta G_{\mathrm{T}}\) are the free energies of the mutated and the original hairpins, T, shown in Figure 3A. To analyze terminator functionality, mutations were introduced into the attenuator structure, which mainly affected conserved positions in the overlap region of the antimetinator and terminator hairpins, H\({}_{2}\) and H\({}_{3}\). The list of mutant strains and the positions of the replaced nucleotides are summarized in Table 1 (see also Figure 3). Calculations of the change in the energies of terminator hairpin formation in the attenuator of the _ilvBNC_ operon under the influence of mutations and experimental data on the effect of the latter on the expression of the _ilvBNC_ operon, AHAS activity, and valine production are given in the next section. Experimental Confirmation of a Computer Model of the Transcriptional Regulation of the C. glutamicum _ilvBNC_ Operon  
We chose a strategy using single substitutions that form or eliminate single noncomplementary pairs in hairpins.