## 3 Results
Figure 3 shows that the conserved pair of tetranucleotides GCCC (positions 76-79) and GGGC (positions 110-113) are involved in terminator hairpin formation, and the boundary GCC trinucleotide (positions 76-78) is also involved in antiterminator hairpin formation by forming complementary bonds with the GGC trinucleotide (positions 36-38). On this basis, strains were created with 11 point mutations in the seven positions. We introduced mutations of three types: (1) at positions 110-112, which are responsible for the stability of only the terminator hairpin in the critical region, (2) at positions 77 and 79, which are involved in complementary interactions of the critical region of both the terminator and antiterminator, and (3) at positions 37 and 73, which are involved in the antiterminator and antiantiterminator but not the terminator. We also made two double mutations by combining mutations from different groups. According to the calculated values of changes in terminator hairpin stability (Table 3), we can expect that the level of valine production will decrease with increasing group number, with double mutations falling into the second group. Deviations from the general trend would characterize the contribution of short-living antianti- and antiterminator secondary structures. \begin{table}
\begin{tabular}{l l l l} \hline \hline \multirow{2}{*}{**Mutant Genotype**} & \multirow{2}{*}{**Valine 1 (g/L)**} & **AHAS Activity, Units/mg Protein 2 ** & \multicolumn{1}{l}{**Terminator**} \\  & & **AHAS Activity, Units/mg Protein 2 ** & \multicolumn{1}{l}{**Free Energy Difference, kcal/mol**} \\ \hline ATCC13869 \(\Delta\)_AponA_ & 0.4 \(\pm\) 0.2 & 13.0 \(\pm\) 1.5 & 0 \\ G111A & 12.2 \(\pm\) 0.5 & 116.2 \(\pm\) 10.0 & 6.56 \\ G112A & 12.1 \(\pm\) 0.5 & 86.9 \(\pm\) 8.5 & 5.56 \\ G110A & 10.7 \(\pm\) 0.5 & 84.5 \(\pm\) 8.5 & 6.49 \\ G11 Overall, the experimental data support the hypothesis and are in reasonably good agreement with each other; the high correlation coefficient (r\({}^{2}\) = 0.86, \(p\) < 0.05, Figure 4B) indicates that variations in valine production in mutant strains are largely due to changes in terminator stability alone. The first group of mutations, G110C, G110T, G110A, G111A, and G112A, weakens the terminator structure by disrupting complementarity with the CCC trinucleotide (positions 77-79) at the base of the hairpin, and increases valine synthesis by an order of magnitude (Table 3). In the second group, C77T, C79T, G37A+C77T, and C77T+G112A, the picture is more complex. Three of them, G37A+C77T, C77T, and C79T, slightly destabilize the terminator by replacing the strong G-C pair with the weaker A-U and G-U pairs, resulting in a level of valine production near the regression line (Figure 4B). The combination of C77T and G112A mutations restore the expression of the _ilvBNC_ operon as in the wild type. The A73G and G37A mutations from the third group do not affect the terminator and thus have a moderate-to-weak effect on valine synthesis (Table 3, Figure 4B). From Figure 4B, we can estimate the effective stabilization of the antiterminator as a result of the A73G mutation as \(-1.5\) kcal/mol in free energy, which is much weaker than the value of \(-6.8\) kcal/mol calculated for the hairpin antiterminator from the thermodynamic parameter tables. Experimental data for measuring the level of expression of the _ilvB_ gene in _C. glutamicum_ strains with simulated mutations in the terminator-antiterminator region are presented in Figure 4. It should be noted that all mutations in the regulatory region did not affect the growth rate of the strains. We observed a high correlation (r\({}^{2}\) = 0.94, \(p\) < \(10^{-3}\)) between the level of valine production and the level of _ilvB_ gene expression (Figure 4C), indicating a key contribution  
Figure 4: Correlation between _ilvB_ gene expression levels, production of valine, and energy change in terminator hairpin in mutant strains. (**A**) The relative level of expression of the _ilvB_ gene in the _C.