Functional complementation between mutations at two distant positions in Escherichia coli RNA polymerase as revealed by second-site suppression

Abstract

Subunit-subunit interactions are critical for the assembly of the core of Escherichia coli RNA polymerase. The mutant α-subunit C131A is unable to complement the temperature-sensitive α-R45C mutant strain, which is defective for binding of the β-subunit. In vitro reconstitution experiments, however, indicate that the α-C131A variant is able to form the intermediate α2β , but is defective in contacting the β'-subunit. We used this α-C131A mutant to isolate a suppressor mutation in the β'-subunit. Genetic and biochemical characterization of the β' 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 β '-subunit, by Asp. However, the crystal structure of the bacterial RNA polymerase indicates that the primary mutation (α-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.