S. Sujatha et al., Functional complementation between mutations at two distant positions in Escherichia coli RNA polymerase as revealed by second-site suppression, MOL G GENET, 264(5), 2001, pp. 531-538
Subunit-subunit interactions are critical for the assembly of the core of E
scherichia coli RNA polymerase. The mutant alpha -subunit C131A is unable t
o complement the temperature-sensitive alpha -R45C mutant strain, which is
defective for binding of the beta -subunit. In vitro reconstitution experim
ents, however, indicate that the alpha -C131A variant is: able to form the
intermediate alpha (2)beta, but is defective in contacting the beta'-subuni
t. We used this alpha -C131A mutant to isolate a suppressor mutation in the
beta'-subunit. Genetic and biochemical characterization of the beta' suppr
essor indicates the allele-specific nature of its effect. Sequence analysis
of the suppressor revealed a single substitution of Gly at position 333, a
n evolutionarily conserved position in the conserved region C of the beta'-
subunit, by Asp. However, the crystal structure of the bacterial RNA polyme
rase indicates that the primary mutation (alpha -C131A) and its suppressor
lie far apart. Thus, we propose that long-range interactions, as in this ca
se, may play an important role in the functional assembly of E. coli RNA po
lymerase.