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

Citation
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
Citations number
45
Categorie Soggetti
Molecular Biology & Genetics
Journal title
MOLECULAR AND GENERAL GENETICS
ISSN journal
00268925 → ACNP
Volume
264
Issue
5
Year of publication
2001
Pages
531 - 538
Database
ISI
SICI code
0026-8925(200101)264:5<531:FCBMAT>2.0.ZU;2-M
Abstract
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.