Sujatha S, Ishihama A, Chatterji D
Centre for Cellular and Molecular Biology, Hyderabad, India.
Mol Gen Genet. 2001 Jan;264(5):531-8. doi: 10.1007/s004380000353.
Subunit-subunit interactions are critical for the assembly of the core of Escherichia coli RNA polymerase. The mutant alpha-subunit C131A is unable to complement the temperature-sensitive alpha-R45C mutant strain, which is defective for binding of the beta-subunit. In vitro reconstitution experiments, however, indicate that the alpha-C131A variant is able to form the intermediate alpha2beta, but is defective in contacting the beta'-subunit. We used this alpha-C131A mutant to isolate a suppressor mutation in the beta'-subunit. Genetic and biochemical characterization of the beta' suppressor indicates the allele-specific nature of its effect. Sequence analysis of the suppressor revealed a single substitution of Gly at position 333, an evolutionarily conserved position in the conserved region C of the beta'-subunit, by Asp. However, the crystal structure of the bacterial RNA polymerase indicates that the primary mutation (alpha-C131A) and its suppressor lie far apart. Thus, we propose that long-range interactions, as in this case, may play an important role in the functional assembly of E. coli RNA polymerase.
亚基-亚基相互作用对于大肠杆菌RNA聚合酶核心的组装至关重要。突变的α亚基C131A无法互补温度敏感型α-R45C突变菌株，该菌株在β亚基结合方面存在缺陷。然而，体外重组实验表明，α-C131A变体能够形成中间产物α2β，但在与β'亚基接触方面存在缺陷。我们利用这种α-C131A突变体分离出β'亚基中的一个抑制突变。β'抑制子的遗传和生化特征表明其效应具有等位基因特异性。对抑制子的序列分析揭示，在β'亚基保守区域C中进化保守的第333位的甘氨酸被天冬氨酸单取代。然而，细菌RNA聚合酶的晶体结构表明，主要突变（α-C131A）及其抑制子相距甚远。因此，我们提出，如在这种情况下的长程相互作用，可能在大肠杆菌RNA聚合酶的功能组装中起重要作用。
